Methods and Compositions for Diagnosis and Prognosis of Renal Injury and Renal Failure

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect one or more of Follistatin-related protein 3, Basigin, Cathepsin B, and Tenascin as diagnostic and prognostic biomarker assays in renal injuries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/735,474, filed Dec. 11, 2017, issued as U.S. Pat. No. 11,506,672; which was filed under 35 U.S.C. § 371 as the U.S. national phase of International Patent Application No. PCT/US2016/037053, filed Jun. 10, 2016, which designated the United States; which claims priority from U.S. Provisional Application Nos. 62/174,456 filed Jun. 11, 2015; 62/174,458 filed Jun. 11, 2015; 62/174,460 filed Jun. 11, 2015; and 62/174,461 filed Jun. 11, 2015, each of which is hereby incorporated in its entirety including all tables, figures, and claims.

SEQUENCE LISTING

This application contains a Sequence Listing which is submitted herewith in electronically readable format. The Sequence Listing file was created on Oct. 27, 2022, is named “AST-8157_US_SeqListing.xml”, and its size is 28.3 kb. The entire contents of the Sequence Listing file are incorporated by reference herein.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

The kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill, New York, pages 1741-1830, which are hereby incorporated by reference in their entirety. Renal disease and/or injury may be acute or chronic. Acute and chronic kidney disease are described as follows (from Current Medical Diagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, which are hereby incorporated by reference in their entirety): “Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances is called azotemia. Chronic renal failure (chronic kidney disease) results from an abnormal loss of renal function over months to years”.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) is an abrupt (typically detected within about 48 hours to 1 week) reduction in glomerular filtration. This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) and non-nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that ARF complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions. ARF may be categorized as prerenal, intrinsic renal, or postrenal in causation. Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities. Major causes of ARF are described in the following table, which is adapted from the Merck Manual, 17^(th) ed., Chapter 222, and which is hereby incorporated by reference in their entirety:

Type Risk Factors Prerenal ECF volume depletion Excessive diuresis, hemorrhage, GI losses, loss of intravascular fluid into the extravascular space (due to ascites, peritonitis, pancreatitis, or burns), loss of skin and mucus membranes, renal salt- and water-wasting states Low cardiac output Cardiomyopathy, MI, cardiac tamponade, pulmonary embolism, pulmonary hypertension, positive-pressure mechanical ventilation Low systemic vascular Septic shock, liver failure, antihypertensive drugs resistance Increased renal vascular NSAIDs, cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis, anesthetics, renal artery obstruction, renal vein thrombosis, sepsis, hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin II receptor blockers arteriolar tone (leading to decreased GFR from reduced glomerular transcapillary pressure, especially in patients with bilateral renal artery stenosis) Intrinsic Renal Acute tubular injury Ischemia (prolonged or severe prerenal state): surgery, hemorrhage, arterial or venous obstruction; Toxins: NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, streptozotocin Acute glomerulonephritis ANCA-associated: Crescentic glomerulonephritis, polyarteritis nodosa, Wegener's granulomatosis; Anti- GBM glomerulonephritis: Goodpasture's syndrome; Immune-complex: Lupus glomerulonephritis, postinfectious glomerulonephritis, cryoglobulinemic glomerulonephritis Acute tubulointerstitial Drug reaction (eg, β-lactams, NSAIDs, sulfonamides, nephritis ciprofloxacin, thiazide diuretics, furosemide, phenytoin, allopurinol, pyelonephritis, papillary necrosis Acute vascular nephropathy Vasculitis, malignant hypertension, thrombotic microangiopathies, scleroderma, atheroembolism Infiltrative diseases Lymphoma, sarcoidosis, leukemia Postrenal Tubular precipitation Uric acid (tumor lysis), sulfonamides, triamterene, acyclovir, indinavir, methotrexate, ethylene glycol ingestion, myeloma protein, myoglobin Ureteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue, fungus ball, edema, malignancy, congenital defects; Extrinsic: Malignancy, retroperitoneal fibrosis, ureteral trauma during surgery or high impact injury Bladder obstruction Mechanical: Benign prostatic hyperplasia, prostate cancer, bladder cancer, urethral strictures, phimosis, paraphimosis, urethral valves, obstructed indwelling urinary catheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuron lesion

In the case of ischemic ARF, the course of the disease may be divided into four phases. During an initiation phase, which lasts hours to days, reduced perfusion of the kidney is evolving into injury. Glomerular ultrafiltration reduces, the flow of filtrate is reduced due to debris within the tubules, and back leakage of filtrate through injured epithelium occurs. Renal injury can be mediated during this phase by reperfusion of the kidney. Initiation is followed by an extension phase which is characterized by continued ischemic injury and inflammation and may involve endothelial damage and vascular congestion. During the maintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs, and glomerular filtration and urine output reaches a minimum. A recovery phase can follow in which the renal epithelium is repaired and GFR gradually recovers. Despite this, the survival rate of subjects with ARF may be as low as about 60%.

Acute kidney injury caused by radiocontrast agents (also called contrast media) and other nephrotoxins such as cyclosporine, antibiotics including aminoglycosides and anticancer drugs such as cisplatin manifests over a period of days to about a week. Contrast induced nephropathy (CIN, which is AKI caused by radiocontrast agents) is thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and from the generation of reactive oxygen species that are directly toxic to renal tubular epithelial cells. CIN classically presents as an acute (onset within 24-48 h) but reversible (peak 3-5 days, resolution within 1 week) rise in blood urea nitrogen and serum creatinine.

A commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days or within a period of hospitalization) elevation of serum creatinine. Although the use of serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications. Traditionally, relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI. However, the recent trend has been towards using smaller serum creatinine rises to define AKI. The relationship between serum creatinine rise, AKI and the associated health risks are reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270, 2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which, with the references listed therein, are hereby incorporated by reference in their entirety. As described in these publications, acute worsening renal function (AKI) and increased risk of death and other detrimental outcomes are now known to be associated with very small increases in serum creatinine. These increases may be determined as a relative (percent) value or a nominal value. Relative increases in serum creatinine as small as 20% from the pre-injury value have been reported to indicate acutely worsening renal function (AKI) and increased health risk, but the more commonly reported value to define AKI and increased health risk is a relative increase of at least 25%. Nominal increases as small as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported to indicate worsening renal function and increased risk of death. Various time periods for the serum creatinine to rise to these threshold values have been used to define AKI, for example, ranging from 2 days, 3 days, 7 days, or a variable period defined as the time the patient is in the hospital or intensive care unit. These studies indicate there is not a particular threshold serum creatinine rise (or time period for the rise) for worsening renal function or AKI, but rather a continuous increase in risk with increasing magnitude of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, hereby incorporated by reference in its entirety) investigated both increases and decreases in serum creatinine. Patients with a mild fall in serum creatinine of −0.1 to −0.3 mg/dL following heart surgery had the lowest mortality rate. Patients with a larger fall in serum creatinine (more than or equal to −0.4 mg/dL) or any increase in serum creatinine had a larger mortality rate. These findings caused the authors to conclude that even very subtle changes in renal function (as detected by small creatinine changes within 48 hours of surgery) seriously effect patient's outcomes. In an effort to reach consensus on a unified classification system for using serum creatinine to define AKI in clinical trials and in clinical practice, Bellomo et al., Crit Care. 8(4):R204-12, 2004, which is hereby incorporated by reference in its entirety, proposes the following classifications for stratifying AKI patients:

-   -   “Risk”: serum creatinine increased 1.5 fold from baseline OR         urine production of <0.5 ml/kg body weight/hr for 6 hours;     -   “Injury”: serum creatinine increased 2.0 fold from baseline OR         urine production <0.5 ml/kg/hr for 12 h;     -   “Failure”: serum creatinine increased 3.0 fold from baseline OR         creatinine >355 μmol/l (with a rise of >44) or urine output         below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours;         And included two clinical outcomes:     -   “Loss”: persistent need for renal replacement therapy for more         than four weeks.     -   “ESRD”: end stage renal disease—the need for dialysis for more         than 3 months.         These criteria are called the RIFLE criteria, which provide a         useful clinical tool to classify renal status. As discussed in         Kellum, Crit. Care Med. 36: S141-45, 2008 and Ricci et al.,         Kidney Int. 73, 538-546, 2008, each hereby incorporated by         reference in its entirety, the RIFLE criteria provide a uniform         definition of AKI which has been validated in numerous studies.

More recently, Mehta et al., Crit. Care 11:R31 (doi:10.1186.cc5713), 2007, hereby incorporated by reference in its entirety, proposes the following similar classifications for stratifying AKI patients, which have been modified from RIFLE:

-   -   “Stage I”:increase in serum creatinine of more than or equal to         0.3 mg/dL (>26.4 μmol/L) or increase to more than or equal to         150% (1.5-fold) from baseline OR urine output less than 0.5         mL/kg per hour for more than 6 hours;     -   “Stage II”:increase in serum creatinine to more than 200%         (>2-fold) from baseline OR urine output less than 0.5 mL/kg per         hour for more than 12 hours;     -   “Stage III”: increase in serum creatinine to more than 300%         (>3-fold) from baseline OR serum creatinine ≥354 μmol/L         accompanied by an acute increase of at least 44 μmol/L OR urine         output less than 0.3 mL/kg per hour for 24 hours or anuria for         12 hours.

The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med. 2006; 7(4):177-197, hereby incorporated by reference in its entirety) uses a serum creatinine rise of 25% to define Contrast induced nephropathy (which is a type of AKI). Although various groups propose slightly different criteria for using serum creatinine to detect AKI, the consensus is that small changes in serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk.

Although serial measurement of serum creatinine over a period of days is an accepted method of detecting and diagnosing AKI and is considered one of the most important tools to evaluate AKI patients, serum creatinine is generally regarded to have several limitations in the diagnosis, assessment and monitoring of AKI patients. The time period for serum creatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considered diagnostic for AKI can be 48 hours or longer depending on the definition used. Since cellular injury in AKI can occur over a period of hours, serum creatinine elevations detected at 48 hours or longer can be a late indicator of injury, and relying on serum creatinine can thus delay diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact kidney status and treatment needs during the most acute phases of AKI when kidney function is changing rapidly. Some patients with AKI will recover fully, some will need dialysis (either short term or long term) and some will have other detrimental outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, it does not differentiate between the causes of AKI (pre-renal, intrinsic renal, post-renal obstruction, atheroembolic, etc) or the category or location of injury in intrinsic renal disease (for example, tubular, glomerular or interstitial in origin). Urine output is similarly limited, Knowing these things can be of vital importance in managing and treating patients with AKI.

These limitations underscore the need for better methods to detect and assess AKI, particularly in the early and subclinical stages, but also in later stages when recovery and repair of the kidney can occur. Furthermore, there is a need to better identify patients who are at risk of having an AKI.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions for evaluating renal function in a subject. As described herein, measurement of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and Tenascin (collectively referred to herein as “kidney injury markers, and individually as a “kidney injury marker”) can be used for diagnosis, prognosis, risk stratification, staging, monitoring, categorizing and determination of further diagnosis and treatment regimens in subjects suffering or at risk of suffering from an injury to renal function, reduced renal function, and/or acute renal failure (also called acute kidney injury).

These kidney injury markers may be used, individually or in panels comprising a plurality of kidney injury markers, for risk stratification (that is, to identify subjects at risk for a future injury to renal function, for future progression to reduced renal function, for future progression to ARF, for future improvement in renal function, etc.); for diagnosis of existing disease (that is, to identify subjects who have suffered an injury to renal function, who have progressed to reduced renal function, who have progressed to ARF, etc.); for monitoring for deterioration or improvement of renal function; and for predicting a future medical outcome, such as improved or worsening renal function, a decreased or increased mortality risk, a decreased or increased risk that a subject will require renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or renal transplantation, a decreased or increased risk that a subject will recover from an injury to renal function, a decreased or increased risk that a subject will recover from ARF, a decreased or increased risk that a subject will progress to end stage renal disease, a decreased or increased risk that a subject will progress to chronic renal failure, a decreased or increased risk that a subject will suffer rejection of a transplanted kidney, etc.

In a first aspect, the present invention relates to methods for evaluating renal status in a subject. These methods comprise performing an assay method that is configured to detect one or more kidney injury markers of the present invention in a body fluid sample obtained from the subject. The assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are then correlated to the renal status of the subject. This correlation to renal status may include correlating the assay result(s) to one or more of risk stratification, diagnosis, prognosis, staging, classifying and monitoring of the subject as described herein. Thus, the present invention utilizes one or more kidney injury markers of the present invention for the evaluation of renal injury.

In certain embodiments, the methods for evaluating renal status described herein are methods for risk stratification of the subject; that is, assigning a likelihood of one or more future changes in renal status to the subject. In these embodiments, the assay result(s) is/are correlated to one or more such future changes. The following are preferred risk stratification embodiments.

In preferred risk stratification embodiments, these methods comprise determining a subject's risk for a future injury to renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a likelihood of such a future injury to renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In other preferred risk stratification embodiments, these methods comprise determining a subject's risk for future reduced renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a likelihood of such reduced renal function. For example, the measured concentrations may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of suffering a future reduced renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of future reduced renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In still other preferred risk stratification embodiments, these methods comprise determining a subject's likelihood for a future improvement in renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a likelihood of such a future improvement in renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold. For a “negative going” kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.

In yet other preferred risk stratification embodiments, these methods comprise determining a subject's risk for progression to ARF, and the result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a likelihood of such progression to ARF. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

And in other preferred risk stratification embodiments, these methods comprise determining a subject's outcome risk, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a likelihood of the occurrence of a clinical outcome related to a renal injury suffered by the subject. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In such risk stratification embodiments, preferably the likelihood or risk assigned is that an event of interest is more or less likely to occur within 180 days of the time at which the body fluid sample is obtained from the subject. In particularly preferred embodiments, the likelihood or risk assigned relates to an event of interest occurring within a shorter time period such as 18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0 hours of the time at which the body fluid sample is obtained from the subject is equivalent to diagnosis of a current condition.

In preferred risk stratification embodiments, the subject is selected for risk stratification based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF. For example, a subject undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery; a subject having pre-existing congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, or sepsis; or a subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin are all preferred subjects for monitoring risks according to the methods described herein. This list is not meant to be limiting. By “pre-existence” in this context is meant that the risk factor exists at the time the body fluid sample is obtained from the subject. In particularly preferred embodiments, a subject is chosen for risk stratification based on an existing diagnosis of injury to renal function, reduced renal function, or ARF.

In other embodiments, the methods for evaluating renal status described herein are methods for diagnosing a renal injury in the subject; that is, assessing whether or not a subject has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are preferred diagnostic embodiments.

In preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of an injury to renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of such an injury. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of reduced renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of an injury causing reduced renal function. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In yet other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of ARF, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of an injury causing ARF. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal replacement therapy, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a need for renal replacement therapy. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal transplantation, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a need for renal transplantation. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other embodiments, the methods for evaluating renal status described herein are methods for monitoring a renal injury in the subject; that is, assessing whether or not renal function is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are preferred monitoring embodiments.

In preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from an injury to renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from reduced renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from acute renal failure, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In other additional preferred monitoring embodiments, these methods comprise monitoring renal status in a subject at risk of an injury to renal function due to the pre-existence of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject having, or at risk of, an injury to renal function for future persistence of acute kidney injury. “Future persistence” as used herein refers to an existing acute renal injury that will continue for a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours. In certain embodiments the subject has an acute kidney injury at the time the sample is obtained. This is not meant to imply that the subject must have an acute kidney injury at the time the sample is obtained, but rather that the subject, upon onset of an acute kidney injury, suffers from an acute kidney injury that will persist. In various embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the future persistence of the acute kidney injury in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a future persistence of acute kidney injury may be assigned to the subject; alternatively, when the measured concentration is below the threshold, a future improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a future persistence of acute kidney injury may be assigned to the subject; alternatively, when the measured concentration is above the threshold, a future improvement of renal function may be assigned to the subject.

In still other embodiments, the methods for evaluating renal status described herein are methods for classifying a renal injury in the subject; that is, determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to a particular class and/or subclass. The following are preferred classification embodiments.

In preferred classification embodiments, these methods comprise determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and/or Tenascin, is/are correlated to the injury classification for the subject. For example, the measured concentration may be compared to a threshold value, and when the measured concentration is above the threshold, a particular classification is assigned; alternatively, when the measured concentration is below the threshold, a different classification may be assigned to the subject.

A variety of methods may be used by the skilled artisan to arrive at a desired threshold value for use in these methods. For example, the threshold value may be determined from a population of normal subjects by selecting a concentration representing the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th) percentile of a kidney injury marker measured in such normal subjects. Alternatively, the threshold value may be determined from a “diseased” population of subjects, e.g., those suffering from an injury or having a predisposition for an injury (e.g., progression to ARF or some other clinical outcome such as death, dialysis, renal transplantation, etc.), by selecting a concentration representing the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th) percentile of a kidney injury marker measured in such subjects. In another alternative, the threshold value may be determined from a prior measurement of a kidney injury marker in the same subject; that is, a temporal change in the level of a kidney injury marker in the subject may be used to assign risk to the subject.

The foregoing discussion is not meant to imply, however, that the kidney injury markers of the present invention must be compared to corresponding individual thresholds. Methods for combining assay results can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, calculating ratios of markers, etc. This list is not meant to be limiting. In these methods, a composite result which is determined by combining individual markers may be treated as if it is itself a marker; that is, a threshold may be determined for the composite result as described herein for individual markers, and the composite result for an individual patient compared to this threshold.

The ability of a particular test to distinguish two populations can be established using ROC analysis. For example, ROC curves established from a “first” subpopulation which is predisposed to one or more future changes in renal status, and a “second” subpopulation which is not so predisposed can be used to calculate a ROC curve, and the area under the curve provides a measure of the quality of the test. Preferably, the tests described herein provide a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.

In certain aspects, the measured concentration of one or more kidney injury markers, or a composite of such markers, may be treated as continuous variables. For example, any particular concentration can be converted into a corresponding probability of a future reduction in renal function for the subject, the occurrence of an injury, a classification, etc. In yet another alternative, a threshold that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into “bins” such as a “first” subpopulation (e.g., which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc.) and a “second” subpopulation which is not so predisposed. A threshold value is selected to separate this first and second population by one or more of the following measures of test accuracy:

-   -   an odds ratio greater than 1, preferably at least about 2 or         more or about 0.5 or less, more preferably at least about 3 or         more or about 0.33 or less, still more preferably at least about         4 or more or about 0.25 or less, even more preferably at least         about 5 or more or about 0.2 or less, and most preferably at         least about 10 or more or about 0.1 or less;     -   a specificity of greater than 0.5, preferably at least about         0.6, more preferably at least about 0.7, still more preferably         at least about 0.8, even more preferably at least about 0.9 and         most preferably at least about 0.95, with a corresponding         sensitivity greater than 0.2, preferably greater than about 0.3,         more preferably greater than about 0.4, still more preferably at         least about 0.5, even more preferably about 0.6, yet more         preferably greater than about 0.7, still more preferably greater         than about 0.8, more preferably greater than about 0.9, and most         preferably greater than about 0.95;     -   a sensitivity of greater than 0.5, preferably at least about         0.6, more preferably at least about 0.7, still more preferably         at least about 0.8, even more preferably at least about 0.9 and         most preferably at least about 0.95, with a corresponding         specificity greater than 0.2, preferably greater than about 0.3,         more preferably greater than about 0.4, still more preferably at         least about 0.5, even more preferably about 0.6, yet more         preferably greater than about 0.7, still more preferably greater         than about 0.8, more preferably greater than about 0.9, and most         preferably greater than about 0.95;     -   at least about 75% sensitivity, combined with at least about 75%         specificity;     -   a positive likelihood ratio (calculated as         sensitivity/(1-specificity)) of greater than 1, at least about         2, more preferably at least about 3, still more preferably at         least about 5, and most preferably at least about 10; or     -   a negative likelihood ratio (calculated as         (1-sensitivity)/specificity) of less than 1, less than or equal         to about 0.5, more preferably less than or equal to about 0.3,         and most preferably less than or equal to about 0.1.         The term “about” in the context of any of the above measurements         refers to +/−5% of a given measurement.

Multiple thresholds may also be used to assess renal status in a subject. For example, a “first” subpopulation which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc., and a “second” subpopulation which is not so predisposed can be combined into a single group. This group is then subdivided into three or more equal parts (known as tertiles, quartiles, quintiles, etc., depending on the number of subdivisions). An odds ratio is assigned to subjects based on which subdivision they fall into. If one considers a tertile, the lowest or highest tertile can be used as a reference for comparison of the other subdivisions. This reference subdivision is assigned an odds ratio of 1. The second tertile is assigned an odds ratio that is relative to that first tertile. That is, someone in the second tertile might be 3 times more likely to suffer one or more future changes in renal status in comparison to someone in the first tertile. The third tertile is also assigned an odds ratio that is relative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodies for use in such assays will specifically bind a full length kidney injury marker of interest, and may also bind one or more polypeptides that are “related” thereto, as that term is defined hereinafter. Numerous immunoassay formats are known to those of skill in the art. Preferred body fluid samples are selected from the group consisting of urine, blood, serum, saliva, tears, and plasma.

The foregoing method steps should not be interpreted to mean that the kidney injury marker assay result(s) is/are used in isolation in the methods described herein. Rather, additional variables or other clinical indicia may be included in the methods described herein. For example, a risk stratification, diagnostic, classification, monitoring, etc. method may combine the assay result(s) with one or more variables measured for the subject selected from the group consisting of demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine), a serum or plasma neutrophil gelatinase (NGAL) concentration, a urine NGAL concentration, a serum or plasma cystatin C concentration, a serum or plasma cardiac troponin concentration, a serum or plasma BNP concentration, a serum or plasma NTproBNP concentration, and a serum or plasma proBNP concentration. Other measures of renal function which may be combined with one or more kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.

When more than one marker is measured, the individual markers may be measured in samples obtained at the same time, or may be determined from samples obtained at different (e.g., an earlier or later) times. The individual markers may also be measured on the same or different body fluid samples. For example, one kidney injury marker may be measured in a serum or plasma sample and another kidney injury marker may be measured in a urine sample. In addition, assignment of a likelihood may combine an individual kidney injury marker assay result with temporal changes in one or more additional variables.

In various related aspects, the present invention also relates to devices and kits for performing the methods described herein. Suitable kits comprise reagents sufficient for performing an assay for at least one of the described kidney injury markers, together with instructions for performing the described threshold comparisons.

In certain embodiments, reagents for performing such assays are provided in an assay device, and such assay devices may be included in such a kit. Preferred reagents can comprise one or more solid phase antibodies, the solid phase antibody comprising antibody that detects the intended biomarker target(s) bound to a solid support. In the case of sandwich immunoassays, such reagents can also include one or more detectably labeled antibodies, the detectably labeled antibody comprising antibody that detects the intended biomarker target(s) bound to a detectable label. Additional optional elements that may be provided as part of an assay device are described hereinafter.

Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, ecl (electrochemical luminescence) labels, metal chelates, colloidal metal particles, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or through the use of a specific binding molecule which itself may be detectable (e.g., a labeled antibody that binds to the second antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Generation of a signal from the signal development element can be performed using various optical, acoustical, and electrochemical methods well known in the art. Examples of detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ellipsometry, etc. In certain of these methods, the solid phase antibody is coupled to a transducer (e.g., a diffraction grating, electrochemical sensor, etc) for generation of a signal, while in others, a signal is generated by a transducer that is spatially separate from the solid phase antibody (e.g., a fluorometer that employs an excitation light source and an optical detector). This list is not meant to be limiting. Antibody-based biosensors may also be employed to determine the presence or amount of analytes that optionally eliminate the need for a labeled molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in subjects suffering or at risk of suffering from injury to renal function, reduced renal function and/or acute renal failure through measurement of one or more kidney injury markers. In various embodiments, a measured concentration of one or more markers selected from the group consisting of Follistatin-related protein 3, Basigin, Cathepsin B, and Tenascin or one or more markers related thereto, and optionally one or more additional kidney injury markers known in the art, are correlated to the renal status of the subject.

For purposes of this document, the following definitions apply:

-   -   As used herein, an “injury to renal function” is an abrupt         (within 14 days, preferably within 7 days, more preferably         within 72 hours, and still more preferably within 48 hours)         measurable reduction in a measure of renal function. Such an         injury may be identified, for example, by a decrease in         glomerular filtration rate or estimated GFR, a reduction in         urine output, an increase in serum creatinine, an increase in         serum cystatin C, a requirement for renal replacement therapy,         etc. “Improvement in Renal Function” is an abrupt (within 14         days, preferably within 7 days, more preferably within 72 hours,         and still more preferably within 48 hours) measurable increase         in a measure of renal function. Preferred methods for measuring         and/or estimating GFR are described hereinafter.     -   As used herein, “reduced renal function” is an abrupt (within 14         days, preferably within 7 days, more preferably within 72 hours,         and still more preferably within 48 hours) reduction in kidney         function identified by an absolute increase in serum creatinine         of greater than or equal to 0.1 mg/dL (≥8.8 μmol/L), a         percentage increase in serum creatinine of greater than or equal         to 20% (1.2-fold from baseline), or a reduction in urine output         (documented oliguria of less than 0.5 ml/kg per hour).     -   As used herein, “acute renal failure” or “ARF” is an abrupt         (within 14 days, preferably within 7 days, more preferably         within 72 hours, and still more preferably within 48 hours)         reduction in kidney function identified by an absolute increase         in serum creatinine of greater than or equal to 0.3 mg/dl (≥26.4         μmol/l), a percentage increase in serum creatinine of greater         than or equal to 50% (1.5-fold from baseline), or a reduction in         urine output (documented oliguria of less than 0.5 ml/kg per         hour for at least 6 hours). This term is synonymous with “acute         kidney injury” or “AKI.”

In this regard, the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such “immunoreactive” polypeptides present in the sample. Expression of biomarkers may also be determined by means other than immunoassays, including protein measurements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting.

As used herein, the term “Follistatin-related protein 3” refers to one or more polypeptides present in a biological sample that are derived from a Follistatin-related protein 3 precursor (human precursor Swiss-Prot entry 095633 (SEQ ID NO: 1)):

        10         20         30         40 MRPGAPGPLW PLPWGALAWA VGFVSSMGSG NPAPGGVCWL         50         60         70         80 QQGQEATCSL VLQTDVTRAE CCASGNIDTA WSNLTHPGNK         90        100        110        120 INLLGFLGLV HCLPCKDSCD GVECGPGKAC RMLGGRPRCE        130        140        150        160 CAPDCSGLPA RLQVCGSDGA TYRDECELRA ARCRGHPDLS        170        180        190        200 VMYRGRCRKS CEHVVCPRPQ SCVVDQTGSA HCVVCRAAPC        210        220        230        240  PVPSSPGQEL CGNNNVTYIS SCHMRQATCF LGRSIGVRHA        250        260 GSCAGTPEEP PGGESAEEEE NFV

The following domains have been identified in Follistatin-related protein 3:

Residues Length Domain ID 1-26 26 signal sequence 27-263 237 Follistatin-related protein 3 1-26 missing in Follistatin-related protein 3 Isoform 2

As used herein, the term “Cathepsin B” refers to one or more polypeptides present in a biological sample that are derived from a Cathepsin B precursor (human precursor Swiss-Prot entry P07858 (SEQ ID NO: 2)):

        10         20         30         40 MWQLWASLCC LLVLANARSR PSFHPLSDEL VNYVNKRNTT         50         60         70         80 WQAGHNFYNV DMSYLKRLCG TFLGGPKPPQ RVMFTEDLKL         90        100        110        120 PASFDAREQW PQCPTIKEIR DQGSCGSCWA FGAVEAISDR        130        140        150        160 ICIHTNAHVS VEVSAEDLLT CCGSMCGDGC NGGYPAEAWN        170        180        190        200 FWTRKGLVSG GLYESHVGCR PYSIPPCEHH VNGSRPPCTG        210        220        230        240 EGDTPKCSKI CEPGYSPTYK QDKHYGYNSY SVSNSEKDIM        250        260        270        280 AEIYKNGPVE GAFSVYSDFL LYKSGVYQHV TGEMMGGHAI        290        300        310        320 RILGWGVENG TPYWLVANSW NTDWGDNGFF KILRGQDHCG        330 IESEVVAGIP RTDQYWEKI

The following domains have been identified in Cathepsin B:

Residues Length Domain ID  1-17 17 signal sequence 18-79 62 activation peptide  80-333 254 Cathepsin B   80-126- 47 Cathepsin B light chain 129-333 205 Cathepsin B heavy chain 334-339 6 propeptide

As used herein, the term “Tenascin” refers to one or more polypeptides present in a biological sample that are derived from a Tenascin precursor (human precursor Swiss-Prot entry P24821 (SEQ ID NO: 3)):

        10         20         30         40 MGAMTQLLAG VFLAFLALAT EGGVLKKVIR HKRQSGVNAT         50         60         70         80 LPEENQPVVF NHVYNIKLPV GSQCSVDLES ASGEKDLAPP         90        100        110        120 SEPSESFQEH TVDGENQIVF THRINIPRRA CGCAAAPDVK        130        140        150        160 ELLSRLEELE NLVSSLREQC TAGAGCCLQP ATGRLDTRPF        170        180        190        200 CSGRGNFSTE GCGCVCEPGW KGPNCSEPEC PGNCHLRGRC        210        220        230        240 IDGQCICDDG FTGEDCSQLA CPSDCNDQGK CVNGVCICFE        250        260        270        280 GYAGADCSRE ICPVPCSEEH GTCVDGLCVC HDGFAGDDCN        290        300        310        320 KPLCLNNCYN RGRCVENECV CDEGFTGEDC SELICPNDCF        330        340        350        360 DRGRCINGTC YCEEGFTGED CGKPTCPHAC HTQGRCEEGQ        370        380        390        400 CVCDEGFAGV DCSEKRCPAD CHNRGRCVDG RCECDDGFTG        410        420        430        440 ADCGELKCPN GCSGHGRCVN GQCVCDEGYT GEDCSQLRCP        450        460        470        480 NDCHSRGRCV EGKCVCEQGF KGYDCSDMSC PNDCHQHGRC        490        500        510        520 VNGMCVCDDG YTGEDCRDRQ CPRDCSNRGL CVDGQCVCED        530        540        550        560 GFTGPDCAEL SCPNDCHGQG RCVNGQCVCH EGFMGKDCKE        570        580        590        600 QRCPSDCHGQ GRCVDGQCIC HEGFTGLDCG QHSCPSDCNN        610        620        630        640 LGQCVSGRCI CNEGYSGEDC SEVSPPKDLV VTEVTEETVN        650        660        670        680 LAWDNEMRVT EYLVVYTPTH EGGLEMQFRV PGDQTSTIIQ        690        700        710        720 ELEPGVEYFI RVFAILENKK SIPVSARVAT YLPAPEGLKF        730        740        750        760 KSIKETSVEV EWDPLDIAFE TWEIIFRNMN KEDEGEITKS        770        780        790        800 LRRPETSYRQ TGLAPGQEYE ISLHIVKNNT RGPGLKRVTT        810        820        830        840 TRLDAPSQIE VKDVTDTTAL ITWFKPLAEI DGIELTYGIK        850        860        870        880 DVPGDRTTID LTEDENQYSI GNLKPDTEYE VSLISRRGDM        890        900        910        920 SSNPAKETFT TGLDAPRNLR RVSQTDNSIT LEWRNGKAAI        930        940        950        960 DSYRIKYAPI SGGDHAEVDV PKSQQATTKT TLTGLRPGTE        970        980        990       1000 YGIGVSAVKE DKESNPATIN AATELDTPKD LQVSETAETS       1010       1020       1030       1040 LTLLWKTPLA KFDRYRLNYS LPTGQWVGVQ LPRNTTSYVL       1050       1060       1070       1080 RGLEPGQEYN VLLTAEKGRH KSKPARVKAS TEQAPELENL       1090       1100       1110       1120 TVTEVGWDGL RLNWTAADQA YEHFIIQVQE ANKVEAARNL       1130       1140       1150       1160 TVPGSLRAVD IPGLKAATPY TVSIYGVIQG YRTPVLSAEA       1170       1180       1190       1200 STGETPNLGE VVVAEVGWDA LKLNWTAPEG AYEYFFIQVQ       1210       1220       1230       1240 EADTVEAAQN LTVPGGLRST DLPGLKAATH YTITIRGVTQ       1250       1260       1270       1280 DFSTTPLSVE VLTEEVPDMG NLTVTEVSWD ALRLNWTTPD       1290       1300       1310       1320 GTYDQFTIQV QEADQVEEAH NLTVPGSLRS MEIPGLRAGT       1330       1340       1350       1360 PYTVTLHGEV RGHSTRPLAV EVVTEDLPQL GDLAVSEVGW       1370       1380       1390       1400 DGLRLNWTAA DNAYEHFVIQ VQEVNKVEAA QNLTLPGSLR       1410       1420       1430       1440 AVDIPGLEAA TPYRVSIYGV IRGYRTPVLS AEASTAKEPE       1450       1460       1470       1480 IGNLNVSDIT PESFNLSWMA TDGIFETFTI EIIDSNRLLE       1490       1500       1510       1520 TVEYNISGAE RTAHISGLPP STDFIVYLSG LAPSIRTKTI       1530       1540       1550       1560 SATATTEALP LLENLTISDI NPYGFTVSWM ASENAFDSFL       1570       1580       1590       1600 VTVVDSGKLL DPQEFTLSGT QRKLELRGLI TGIGYEVMVS       1610       1620       1630       1640 GFTQGHQTKP LRAEIVTEAE PEVDNLLVSD ATPDGFRLSW       1650       1660       1670       1680 TADEGVFDNF VLKIRDTKKQ SEPLEITLLA PERTRDITGL       1690       1700       1710       1720 REATEYEIEL YGISKGRRSQ TVSAIATTAM GSPKEVIFSD       1730       1740       1750       1760 ITENSATVSW RAPTAQVESF RITYVPITGG TPSMVTVDGT       1770       1780       1790       1800 KTQTRLVKLI PGVEYLVSII AMKGFEESEP VSGSFTTALD       1810       1820       1830       1840 GPSGLVTANI TDSEALARWQ PAIATVDSYV ISYTGEKVPE       1850       1860       1870       1880 ITRTVSGNTV EYALTDLEPA TEYTLRIFAE KGPQKSSTIT       1890       1900       1910       1920 AKFTTDLDSP RDLTATEVQS ETALLTWRPP RASVTGYLLV       1930       1940       1950       1960 YESVDGTVKE VIVGPDTTSY SLADLSPSTH YTAKIQALNG       1970       1980       1990       2000 PLRSNMIQTI FTTIGLLYPF PKDCSQAMLN GDTTSGLYTI       2010       2020       2030       2040 YLNGDKAEAL EVFCDMTSDG GGWIVFLRRK NGRENFYQNW       2050       2060       2070       2080 KAYAAGFGDR REEFWLGLDN LNKITAQGQY ELRVDLRDHG       2090       2100       2110       2120 ETAFAVYDKF SVGDAKTRYK LKVEGYSGTA GDSMAYHNGR       2130       2140       2150       2160 SFSTFDKDTD SAITNCALSY KGAFWYRNCH RVNLMGRYGD       2170       2180       2190       2200 NNHSQGVNWF HWKGHEHSIQ FAEMKLRPSN FRNLEGRRKR A

The following domains have been identified in Tenascin:

Residues Length Domain ID  1-22 22 signal sequence  23-2201 2179 Tenascin 1072-1435 missing in Tenascin Isoform 2 1527-1617 missing in Tenascin Isoform 2 1072-1435 missing in Tenascin isoform 3 1527-1617 missing in Tenascin isoform 4 1072-1617 missing in Tenascin isoform 5 1072-1708 missing in Tenascin isoform 6

As used herein, the term “Basigin” refers to one or more polypeptides present in a biological sample that are derived from a Basigin precursor (human precursor Swiss-Prot entry P35613 (SEQ ID NO: 4)):

        10         20         30         40 MAAALFVLLG FALLGTHGAS GAAGFVQAPL SQQRWVGGSV         50         60         70         80 ELHCEAVGSP VPEIQWWFEG QGPNDTCSQL WDGARLDRVH         90        100        110        120 IHATYHQHAA STISIDTLVE EDTGTYECRA SNDPDRNHLT        130        140        150        160 RAPRVKWVRA QAVVLVLEPG TVFTTVEDLG SKILLTCSLN        170        180        190        200 DSATEVTGHR WLKGGVVLKE DALPGQKTEF KVDSDDQWGE        210        220        230        240 YSCVFLPEPM GTANIQLHGP PRVKAVKSSE HINEGETAML        250        260        270        280 VCKSESVPPV TDWAWYKITD SEDKALMNGS ESRFFVSSSQ        290        300        310        320 GRSELHIENL NMEADPGQYR CNGTSSKGSD QAIITLRVRS        330        340        350        360 HLAALWPFLG IVAEVLVLVT IIFIYEKRRK PEDVLDDDDA        370        380 GSAPLKSSGQ HQNDKGKNVR QRNSS

The following domains have been identified in Basigin:

Residues Length Domain ID 1-21 21 signal sequence 22-385 364 Basigin 24-139 missing in Basigin Isoform 2  1-209 missing in Basigin Isoform 3 12-191 missing in Basigin Isoform 4 1-11 → MKQSDASPQER (SEQ ID NO: 5) in Basigin Isoform 4

As used herein, the term “relating a signal to the presence or amount” of an analyte reflects this understanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is “configured to detect” an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte. Because an antibody epitope is on the order of 8 amino acids, an immunoassay configured to detect a marker of interest will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay.

The term “related marker” as used herein with regard to a biomarker such as one of the kidney injury markers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers. The term also refers to one or more polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.

The term “positive going” marker as that term is used herein refer to a marker that is determined to be elevated in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition. The term “negative going” marker as that term is used herein refer to a marker that is determined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.

The term “subject” as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably “patients,” which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement used to evaluate the kidney for preexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability (“a likelihood”) of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, “diagnosis” includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed. That such a diagnosis is “determined” is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) that a given course or outcome will occur. A level or a change in level of a prognostic indicator, which in turn is associated with an increased probability of morbidity (e.g., worsening renal function, future ARF, or death) is referred to as being “indicative of an increased likelihood” of an adverse outcome in a patient.

Marker Assays

In general, immunoassays involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein “blotting” methods. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among the immunoassay analyzers that are capable of performing immunoassays. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.

Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysis of the described kidney injury markers. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that a kidney injury marker. The kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein. Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte. Preferably, an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies that bind a kidney injury marker. Most preferably each of the antibodies are monoclonal antibodies. The instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”

Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker of the present invention. The term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, Preferred antibodies bind with affinities of at least about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹ M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹² M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is the dissociation rate constant, K_(on) is the association rate constant and K_(d) is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n=number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

The term “epitope” refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.

The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.

Assay Correlations

The term “correlating” as used herein in reference to the use of biomarkers refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5^(th) percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior “baseline” result is used to monitor for temporal changes in a biomarker level.

Population studies may also be used to select a decision threshold. Reciever Operating Characteristic (“ROC”) arose from the field of signal dectection therory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold able to best distinguish a “diseased” subpopulation from a “nondiseased” subpopulation. A false positive in this case occurs when the person tests positive, but actually does not have the disease. A false negative, on the other hand, occurs when the person tests negative, suggesting they are healthy, when they actually do have the disease. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1−specificity, the ROC graph is sometimes called the sensitivity vs (1−specificity) plot. A perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5. A threshold is selected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and “nondiseased” is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (“AUC”) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1-specificity)) of greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1-sensitivity)/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1

Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention. These include other biomarkers related to renal status. Examples include the following, which recite the common biomarker name, followed by the Swiss-Prot entry number for that biomarker or its parent: Actin (P68133); Adenosine deaminase binding protein (DPP4, P27487); Alpha-1-acid glycoprotein 1 (P02763); Alpha-1-microglobulin (P02760); Albumin (P02768); Angiotensinogenase (Renin, P00797); Nidogen-1 (P07355); Beta-glucuronidase (P08236); B-2-microglobulin (P61769); Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta (S100-beta, P04271); Carbonic anhydrase 9 (Q16790); Casein Kinase 2 (P68400); Clusterin (P10909); Complement C3 (P01024); Cysteine-rich protein (CYR61, 000622); Cytochrome C (P99999); Epidermal growth factor (EGF, P01133); Endothelin-1 (P05305); Exosomal Fetuin-A (P02765); Fatty acid-binding protein, heart (FABP3, P05413); Fatty acid-binding protein, liver (P07148); Ferritin (light chain, P02792; heavy chain P02794); Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCL1, (P09341); Growth Hormone (P01241); Hepatocyte growth factor (P14210); Insulin-like growth factor I (P05019); Immunoglobulin G; Immunoglobulin Light Chains (Kappa and Lambda); Interferon gamma (P01308); Lysozyme (P61626); Interleukin-1alpha (P01583); Interleukin-2 (P60568); Interleukin-4 (P05112); Interleukin-9 (P15248); Interleukin-12p40 (P29460); Interleukin-13 (P35225); Interleukin-16 (Q14005); L1 cell adhesion molecule (P32004); Lactate dehydrogenase (P00338); Leucine Aminopeptidase (P28838); Meprin A-alpha subunit (Q16819); Meprin A-beta subunit (Q16820); Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2 (P08253); MMP-9 (P14780); Netrin-1 (O95631); Neutral endopeptidase (P08473); Osteopontin (O14788); Renal papillary antigen 1 (RPA1); Renal papillary antigen 2 (RPA2); Retinol binding protein (P09455); Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum Amyloid P Component (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764); Spermidine/spermine N1-acetyltransferase (P21673); TGF-Beta1 (P01137); Transferrin (P02787); Trefoil factor 3 (TFF3, Q07654); Toll-Like protein 4 (O00206); Total protein; Tubulointerstitial nephritis antigen (Q9UJW2); Uromodulin (Tamm-Horsfall protein, P07911).

For purposes of risk stratification, Adiponectin (Q15848); Alkaline phosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937); Cystatin C (P01034); 8 subunit of FIFO ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa (alpha-glutathione-S-transferase, P08263); GSTpi (Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1 (P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itm1, P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18 (Q14116); IP-10 (10 kDa interferon-gamma-induced protein, P02778); IRPR (IFRD1, 000458); Isovaleryl-CoA dehydrogenase (IVD, P26440); I-TAC/CXCL11 (O14625); Keratin 19 (P08727); Kim-1 (Hepatitis A virus cellular receptor 1, 043656); L-arginine:glycine amidinotransferase (P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500); MIG (Gamma-interferon-induced monokine Q07325); MIP-la (P10147); MIP-3a (P78556); MIP-1beta (P13236); MIP-1d (Q16663); NAG (N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter (OCT2, O15244); Osteoprotegerin (O14788); P8 protein (O60356); Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP (1-98) (P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta (P50395); Renal kallikrein (P06870); RT1.B-1 (alpha) chain of the integral membrane protein (Q5Y7A8); soluble tumor necrosis factor receptor superfamily member 1A (sTNFR-I, P19438); soluble tumor necrosis factor receptor superfamily member 1B (sTNFR-II, P20333); Tissue inhibitor of metalloproteinases 3 (TIMP-3, P35625); uPAR (Q03405) may be combined with the kidney injury marker assay result(s) of the present invention.

Other clinical indicia which may be combined with the kidney injury marker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total protein measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal papillary antigen 1 (RPA1) measurement; a renal papillary antigen 2 (RPA2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine). Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.

Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.

Diagnosis of Acute Renal Failure

As noted above, the terms “acute renal (or kidney) injury” and “acute renal (or kidney) failure” as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:

${GFR} = \frac{{Urine}{Concentration} \times {Urine}{Flow}}{{Plasma}{Concentration}}$

By normalizing the GFR to the body surface area, a GFR of approximately ml/min per 1.73 m² can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.

There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.

Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (U_(Cr)), urine flow rate (V), and creatinine's plasma concentration (P_(Cr)) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (U_(Cr)×V) divided by its plasma concentration. This is commonly represented mathematically as:

$C_{Cr} = \frac{U_{Cr} \times V}{P_{Cr}}$

Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:

$C_{Cr} = \frac{U_{Cr} \times 24 - {hour}{volume}}{P_{Cr} \times 24 \times 60{mins}}$

To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:

$C_{{Cr} - {corrected}} = \frac{C_{Cr} \times 1.73}{BSA}$

The accuracy of a creatinine clearance measurement (even when collection is complete) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.

For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are provided, minor modifications of the RIFLE urine output criteria have been described. For example, Bagshaw et al., Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure).

Selecting a Treatment Regimen

Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, NJ, 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.

ARF can often be prevented by proper maintenance of normal fluid balance, blood volume, and BP during and after major surgery; by adequate isotonic NaCl infusions in patients with severe burns; and by prompt transfusion in hemorrhagic hypotension. When a vasopressor drug is required, dopamine 1 to 3 μg/kg/min IV may augment renal blood flow, and urine output, but there is no clinical evidence that ARF is averted. In incipient ARF, furosemide with mannitol or dopamine may, reestablish normal urine flow or convert oliguric to nonoliguric ARF, but little evidence exists that the mortality rate is reduced.

Dehydration and ECF depletion should be avoided in patients requiring cholecystography or in patients with renal insufficiency requiring urography, particularly those with multiple myeloma. Urography and angiography should be avoided in any patient with renal insufficiency because of the high incidence of renal deterioration. The higher incidence in elderly patients correlates with the natural fall in GFR with aging. Before cytolytic therapy in patients with certain neoplastic diseases (e.g., lymphoma, leukemia), treatment with allopurinol should be considered along with alkalinizing the urine (oral sodium bicarbonate or acetazolamide) and increasing urine flow with increased oral or IV fluids to reduce urate crystalluria.

Dialysis improves fluid and electrolyte imbalances and allows adequate nutrition. No consensus exists as to when to begin dialysis, how frequently to perform it, or whether it even improves recovery or survival. However, hemodialysis with biocompatible membranes (e.g., polysulfone, polyacrylonitrile, polymethylmethacrylate) rather than cuprophane membranes may improve the recovery of renal function and reduce the mortality rate.

ARF should be managed without dialysis only when dialysis is unavailable or the course of ARF is uncomplicated and has existed <5 days. All renally excreted substances (e.g., digoxin, some antibiotics) must be adjusted. Water intake should be restricted to a volume equal to urine output plus measured extrarenal losses plus 500 mL/day for insensible loss. Water intake can be further modified to keep the serum Na concentration in the normal range. Body weight is an indication of fluid intake; weight loss of up to 0.5 kg/day is expected in patients not meeting basal caloric needs, and any weight gain must be attributed to excess fluid. Na and K intake is minimized, except when prior deficiencies or GI losses occurred. To reduce nitrogen loss; oral or IV administration of essential amino acids with glucose or a highly concentrated carbohydrate has been advocated, but risks include fluid overload, hyperosmolality, and infection. Ca salts (carbonate, acetate) before meals help maintain serum phosphorus at <5.5 mg/dL (<1.78 mmol/L). To help maintain serum K at <6 mmol/L in the absence of dialysis, a cation-exchange resin, sodium polystyrene sulfonate, is given 15 g po or rectally 1 to 4 times/day as a suspension in water or in a syrup (e.g., 70% sorbitol). An indwelling bladder catheter is rarely needed and should be used only when necessary because of an increased risk of UTI and urosepsis.

When obstruction is relieved, polyuria characterized by excretion of large amounts of Na, K, Mg, and other solutes may result. Self-limited hypokalemia; hyponatremia, hypernatremia, hypomagnesemia, or marked contraction of ECF volume with peripheral vascular collapse can occur. In many patients, a brisk diuresis after relief of obstruction is a physiologic response to the expansion of ECF during obstruction and does not compromise volume status. Overzealous administration of salt and water after relief of obstruction can prolong the diuresis.

In the postoliguric phase, close attention to fluid and electrolyte balance is mandatory to present potentially serious or lethal disturbances in extracellular volume, osmolality, acid-base balance, and K balance.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Example 1: Contrast-Induced Nephropathy Sample Collection

The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after receiving intravascular contrast media. Approximately 250 adults undergoing radiographic/angiographic procedures involving intravascular administration of iodinated contrast media are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

-   -   males and females 18 years of age or older;     -   undergoing a radiographic/angiographic procedure (such as a CT         scan or coronary intervention) involving the intravascular         administration of contrast media;     -   expected to be hospitalized for at least 48 hours after contrast         administration.     -   able and willing to provide written informed consent for study         participation and to comply with all study procedures.

Exclusion Criteria

-   -   renal transplant recipients;     -   acutely worsening renal function prior to the contrast         procedure;     -   already receiving dialysis (either acute or chronic) or in         imminent need of dialysis at enrollment;     -   expected to undergo a major surgical procedure (such as         involving cardiopulmonary bypass) or an additional imaging         procedure with contrast media with significant risk for further         renal insult within the 48 hrs following contrast         administration;     -   participation in an interventional clinical study with an         experimental therapy within the previous 30 days;     -   known infection with human immunodeficiency virus (HIV) or a         hepatitis virus.

Immediately prior to the first contrast administration (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL) and a urine sample (10 mL) are collected from each patient. Blood and urine samples are then collected at 4 (±0.5), 8 (±1), 24 (±2) 48 (±2), and 72 (±2) hrs following the last administration of contrast media during the index contrast procedure. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.

Serum creatinine is assessed at the site immediately prior to the first contrast administration (after any pre-procedure hydration) and at 4 (±0.5), 8 (±1), 24 (±2) and 48 (±2)), and 72 (±2) hours following the last administration of contrast (ideally at the same time as the study samples are obtained). In addition, each patient's status is evaluated through day 30 with regard to additional serum and urine creatinine measurements, a need for dialysis, hospitalization status, and adverse clinical outcomes (including mortality).

Prior to contrast administration, each patient is assigned a risk based on the following assessment: systolic blood pressure <80 mm Hg=5 points; intra-arterial balloon pump=5 points; congestive heart failure (Class III-IV or history of pulmonary edema)=5 points; age >75 yrs=4 points; hematocrit level <39% for men, <35% for women=3 points; diabetes=3 points; contrast media volume=1 point for each 100 mL; serum creatinine level >1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m²=2 points, 20-40 mL/min/1.73 m²=4 points, <20 mL/min/1.73 m²=6 points. The risks assigned are as follows: risk for CIN and dialysis: 5 or less total points=risk of CIN—7.5%, risk of dialysis—0.04%; 6-10 total points=risk of CIN—14%, risk of dialysis—11-16 total points=risk of CIN—26.1%, risk of dialysis—1.09%; >16 total points=risk of CIN—57.3%, risk of dialysis—12.8%.

Example 2: Cardiac Surgery Sample Collection

The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after undergoing cardiovascular surgery, a procedure known to be potentially damaging to kidney function. Approximately 900 adults undergoing such surgery are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

-   -   males and females 18 years of age or older;     -   undergoing cardiovascular surgery;     -   Toronto/Ottawa Predictive Risk Index for Renal Replacement risk         score of at least 2 (Wijeysundera et al., JAMA 297: 1801-9,         2007); and     -   able and willing to provide written informed consent for study         participation and to comply with all study procedures.

Exclusion Criteria

-   -   known pregnancy;     -   previous renal transplantation;     -   acutely worsening renal function prior to enrollment (e.g., any         category of RIFLE criteria);     -   already receiving dialysis (either acute or chronic) or in         imminent need of dialysis at enrollment;     -   currently enrolled in another clinical study or expected to be         enrolled in another clinical study within 7 days of cardiac         surgery that involves drug infusion or a therapeutic         intervention for AKI;     -   known infection with human immunodeficiency virus (HIV) or a         hepatitis virus.

Within 3 hours prior to the first incision (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3 mL), and a urine sample (35 mL) are collected from each patient. Blood and urine samples are then collected at 3 (±0.5), 6 (±0.5), 12 (±1), 24 (±2) and 48 (±2) hrs following the procedure and then daily on days 3 through 7 if the subject remains in the hospital. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.

Example 3: Acutely Ill Subject Sample Collection

The objective of this study is to collect samples from acutely ill patients. Approximately 900 adults expected to be in the ICU for at least 48 hours will be enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

-   -   males and females 18 years of age or older;     -   Study population 1: approximately 300 patients that have at         least one of:     -   shock (SBP<90 mmHg and/or need for vasopressor support to         maintain MAP>60 mmHg and/or documented drop in SBP of at least         40 mmHg); and     -   sepsis;     -   Study population 2: approximately 300 patients that have at         least one of:     -   IV antibiotics ordered in computerized physician order entry         (CPOE) within 24 hours of enrollment;     -   contrast media exposure within 24 hours of enrollment;     -   increased Intra-Abdominal Pressure with acute decompensated         heart failure; and     -   severe trauma as the primary reason for ICU admission and likely         to be hospitalized in the ICU for 48 hours after enrollment;     -   Study population 3: approximately 300 patients     -   expected to be hospitalized through acute care setting (ICU or         ED) with a known risk factor for acute renal injury (e.g.         sepsis, hypotension/shock (Shock=systolic BP<90 mmHg and/or the         need for vasopressor support to maintain a MAP>60 mmHg and/or a         documented drop in SBP>40 mmHg), major trauma, hemorrhage, or         major surgery); and/or expected to be hospitalized to the ICU         for at least 24 hours after enrollment.

Exclusion Criteria

-   -   known pregnancy;     -   institutionalized individuals;     -   previous renal transplantation;     -   known acutely worsening renal function prior to enrollment         (e.g., any category of RIFLE criteria);     -   received dialysis (either acute or chronic) within 5 days prior         to enrollment or in imminent need of dialysis at the time of         enrollment;     -   known infection with human immunodeficiency virus (HIV) or a         hepatitis virus;     -   meets only the SBP<90 mmHg inclusion criterion set forth above,         and does not have shock in the attending physician's or         principal investigator's opinion.

After providing informed consent, an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-30 mL) are collected from each patient. Blood and urine samples are then collected at 4 (±0.5) and 8 (±1) hours after contrast administration (if applicable); at 12 (±1), 24 (±2), and 48 (±2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.

Example 4. Immunoassay Format

Analytes are measured using standard sandwich enzyme immunoassay techniques. A first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate. Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution comprising tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte present in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards. Units for Follistatin-related protein 3, Basigin, and Tenascin reported herein are pg/mL; units for Cathepsin B are ng/mL.

Example 5. Apparently Healthy Donor and Chronic Disease Patient Samples

Human urine samples from donors with no known chronic or acute disease (“Apparently Healthy Donors”) were purchased from two vendors (Golden West Biologicals, Inc., 27625 Commerce Center Dr., Temecula, CA 92590 and Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, VA 23454). The urine samples were shipped and stored frozen at less than −20° C. The vendors supplied demographic information for the individual donors including gender, race (Black/White), smoking status and age.

Human urine samples from donors with various chronic diseases (“Chronic Disease Patients”) including congestive heart failure, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, diabetes mellitus and hypertension were purchased from Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, VA 23454. The urine samples were shipped and stored frozen at less than −20 degrees centigrade. The vendor provided a case report form for each individual donor with age, gender, race (Black/White), smoking status and alcohol use, height, weight, chronic disease(s) diagnosis, current medications and previous surgeries.

Example 6. Use of Follistatin-Related Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Follistatin-related protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 6.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 36900 64700 36400 64500 36400 64500 Average 43900 86000 44100 85700 44100 85700 Stdev 21100 118000 22600 118000 22600 118000 p (t-test) 0.29 0.32 0.32 Min 24900 10500 24900 10500 24900 10500 Max 89300 1230000 89300 1230000 89300 1230000 n (Patient) 9 141 8 142 8 142 sCr only Median 39100 65900 37800 65800 37800 65800 Average 83200 83500 85400 83200 85400 83200 Stdev 154000 110000 158000 109000 158000 109000 p (t-test) 0.99 0.94 0.94 Min 10500 15100 10500 15100 10500 15100 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 18 132 17 133 17 133 UO only Median 47500 77400 47700 74000 47500 73900 Average 58600 102000 59000 100000 58700 99200 Stdev 42400 145000 42900 143000 43700 141000 p (t-test) 0.023 0.031 0.037 Min 15100 10500 15100 10500 15100 10500 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 63 86 60 89 57 92 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.68 0.70 0.74 0.68 0.69 0.74 0.68 0.70 SE 0.070 0.060 0.042 0.075 0.062 0.043 0.075 0.062 0.043 p Value 5.3E−4 0.0025 1.2E−6 0.0013 0.0036 4.9E−6 0.0013 0.0036 4.4E−6 nCohort Recovered 9 18 63 8 17 60 8 17 57 nCohort Non-recovered 141 132 86 142 133 89 142 133 92 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 77% 79% 86% 77% 79% 85% 77% 79% 85% Specificity 67% 56% 40% 75% 59% 40% 75% 59% 40% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 52% 53% 64% 51% 53% 63% 51% 53% 63% Specificity 78% 72% 68% 75% 71% 68% 75% 71% 70% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 27% 27% 36% 27% 26% 36% 27% 26% 36% Specificity 100%  83% 89% 100%  82% 90% 100%  82% 91% OR Quartile 2 6.81 4.64 4.06 10.3 5.36 3.90 10.3 5.36 3.77 p Value 0.0091 0.0031 5.3E−4 0.0055 0.0018 6.6E−4 0.0055 0.0018 8.2E−4 Lower limit of 95% CI 1.61 1.68 1.84 1.98 1.87 1.78 1.98 1.87 1.73 Upper limit of 95% CI 28.8 12.9 8.95 53.6 15.3 8.53 53.6 15.3 8.20 OR Quartile 3 3.76 2.94 3.81 3.17 2.67 3.66 3.17 2.67 4.01 p Value 0.11 0.052 1.4E−4 0.17 0.080 2.4E−4 0.17 0.080 1.2E−4 Lower limit of 95% CI 0.754 0.990 1.91 0.619 0.890 1.83 0.619 0.890 1.98 Upper limit of 95% CI 18.7 8.70 7.60 16.3 7.99 7.33 16.3 7.99 8.15 OR Quartile 4 7.07 1.80 4.51 6.26 1.67 5.05 6.26 1.67 5.82 p Value 0.18 0.37 0.0010 0.21 0.44 8.1E−4 0.21 0.44 6.5E−4 Lower limit of 95% CI 0.402 0.492 1.83 0.353 0.452 1.96 0.353 0.452 2.11 Upper limit of 95% CI 124 6.61 11.1 111 6.15 13.0 111 6.15 16.0

TABLE 6.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 35900 68300 36000 67200 36100 66600 Average 49400 89000 51500 87800 52800 87400 Stdev 52400 122000 56500 120000 58000 120000 p (t-test) 0.14 0.21 0.25 Min 10500 15100 10500 15100 10500 15100 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 21 129 18 132 17 133 sCr only Median 37400 68400 37400 68100 37800 67900 Average 74800 85400 77500 84700 79400 84300 Stdev 130000 112000 134000 111000 137000 111000 p (t-test) 0.66 0.77 0.85 Min 10500 15100 10500 15100 10500 15100 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 28 122 26 124 25 125 UO only Median 49100 81200 49500 74000 48800 74000 Average 59200 108000 59800 105000 59400 104000 Stdev 39700 155000 40600 151000 41000 149000 p (t-test) 0.011 0.021 0.022 Min 10500 21500 10500 21500 10500 21500 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 71 74 66 79 64 81 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.78 0.72 0.71 0.76 0.71 0.69 0.75 0.70 0.70 SE 0.046 0.048 0.043 0.051 0.050 0.043 0.054 0.051 0.043 p Value 1.0E−9 2.7E−6 7.2E−7 3.0E−7 1.6E−5 1.2E−5 3.6E−6 8.3E−5 5.2E−6 nCohort Recovered 21 28 71 18 26 66 17 25 64 nCohort Non-recovered 129 122 74 132 124 79 133 125 81 Cutoff Quartile 2 40800 40800 41800 40800 40800 41800 40800 40800 41800 Sensitivity 81% 82% 86% 80% 81% 85% 80% 81% 85% Specificity 67% 57% 37% 67% 58% 36% 65% 56% 38% Cutoff Quartile 3 63600 63600 64300 63600 63600 64300 63600 63600 64300 Sensitivity 56% 57% 65% 55% 56% 63% 54% 55% 63% Specificity 86% 79% 65% 83% 77% 65% 82% 76% 66% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 29% 28% 38% 28% 27% 37% 28% 27% 37% Specificity 95% 86% 87% 94% 85% 88% 94% 84% 89% OR Quartile 2 8.75 6.06 3.70 8.15 5.99 3.19 7.20 5.36 3.45 p Value 2.6E−5 5.9E−5 0.0018 1.2E−4 9.7E−5 0.0041 3.4E−4 2.8E−4 0.0023 Lower limit of 95% CI 3.19 2.52 1.62 2.80 2.43 1.44 2.44 2.16 1.56 Upper limit of 95% CI 24.0 14.6 8.42 23.8 14.7 7.05 21.2 13.3 7.64 OR Quartile 3 7.58 4.77 3.40 6.00 4.18 3.22 5.51 3.90 3.25 p Value 0.0018 0.0016 4.4E−4 0.0063 0.0042 7.7E−4 0.0097 0.0066 7.6E−4 Lower limit of 95% CI 2.13 1.81 1.72 1.66 1.57 1.63 1.51 1.46 1.64 Upper limit of 95% CI 27.0 12.6 6.72 21.7 11.1 6.38 20.1 10.4 6.44 OR Quartile 4 8.04 2.32 4.19 6.62 2.08 4.20 6.17 1.96 4.79 p Value 0.046 0.14 8.5E−4 0.071 0.21 0.0012 0.083 0.25 7.0E−4 Lower limit of 95% CI 1.04 0.749 1.81 0.850 0.667 1.76 0.789 0.628 1.94 Upper limit of 95% CI 62.1 7.18 9.74 51.5 6.47 10.0 48.2 6.13 11.8

TABLE 6.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 42000 68700 43700 68400 43600 68600 Average 56500 91300 57900 90400 56600 89900 Stdev 49700 127000 51100 126000 53200 124000 p (t-test) 0.13 0.17 0.18 Min 10500 15100 10500 15100 10500 15100 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 32 117 30 119 27 122 sCr only Median 42700 69300 43600 68700 43600 68700 Average 74500 87400 75700 86900 75700 86900 Stdev 113000 117000 114000 117000 114000 117000 p (t-test) 0.56 0.61 0.61 Min 10500 15100 10500 15100 10500 15100 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 38 110 37 111 37 111 UO only Median 54200 73800 55100 72700 55100 72700 Average 62900 109000 62900 107000 63200 103000 Stdev 33800 161000 34300 158000 35100 152000 p (t-test) 0.026 0.034 0.056 Min 15100 23300 15100 23300 15100 23300 Max 181000 1230000 181000 1230000 181000 1230000 n (Patient) 64 69 61 72 55 78 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.68 0.66 0.69 0.67 0.66 0.71 0.67 0.64 SE 0.047 0.047 0.047 0.049 0.048 0.047 0.049 0.048 0.048 p Value 1.1E−5 1.7E−4 5.5E−4 1.1E−4 5.5E−4 7.7E−4 1.8E−5 5.5E−4 0.0026 nCohort Recovered 32 38 64 30 37 61 27 37 55 nCohort Non-recovered 117 110 69 119 111 72 122 111 78 Cutoff Quartile 2 41800 41500 43900 41800 41500 43900 41800 41500 43900 Sensitivity 82% 82% 83% 81% 81% 82% 80% 81% 79% Specificity 50% 45% 33% 47% 43% 33% 48% 43% 31% Cutoff Quartile 3 63900 64100 66600 63900 64100 66600 63900 64100 66600 Sensitivity 58% 58% 61% 57% 58% 60% 57% 58% 59% Specificity 78% 74% 61% 77% 73% 61% 81% 73% 62% Cutoff Quartile 4 91300 91400 93500 91300 91400 93500 91300 91400 93500 Sensitivity 30% 28% 35% 29% 28% 35% 29% 28% 32% Specificity 91% 84% 84% 90% 84% 85% 89% 84% 84% OR Quartile 2 4.57 3.64 2.32 3.65 3.27 2.21 3.79 3.27 1.73 p Value 3.8E−4 0.0016 0.042 0.0028 0.0040 0.053 0.0029 0.0040 0.17 Lower limit of 95% CI 1.98 1.63 1.03 1.56 1.46 0.991 1.58 1.46 0.784 Upper limit of 95% CI 10.6 8.13 5.23 8.54 7.31 4.95 9.11 7.31 3.83 OR Quartile 3 4.96 3.90 2.43 4.38 3.68 2.29 5.92 3.68 2.33 p Value 6.1E−4 0.0011 0.013 0.0017 0.0018 0.020 7.6E−4 0.0018 0.019 Lower limit of 95% CI 1.98 1.72 1.21 1.74 1.62 1.14 2.10 1.62 1.15 Upper limit of 95% CI 12.4 8.80 4.87 11.0 8.33 4.59 16.7 8.33 4.72 OR Quartile 4 4.13 2.09 2.88 3.75 2.00 3.07 3.22 2.00 2.41 p Value 0.027 0.13 0.013 0.039 0.16 0.010 0.070 0.16 0.044 Lower limit of 95% CI 1.18 0.797 1.25 1.07 0.761 1.30 0.910 0.761 1.02 Upper limit of 95% CI 14.4 5.50 6.65 13.2 5.27 7.25 11.4 5.27 5.69

TABLE 6.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 44700 70200 43900 70500 43600 70500 Average 59300 94800 58100 93500 58000 92900 Stdev 46200 133000 48800 130000 50200 129000 p (t-test) 0.11 0.13 0.14 Min 10500 15100 10500 15100 10500 15100 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 38 106 33 111 31 113 sCr only Median 45400 70500 45400 70500 45400 70500 Average 73600 90800 73600 90800 74300 90100 Stdev 104000 123000 104000 123000 106000 122000 p (t-test) 0.42 0.42 0.46 Min 10500 15100 10500 15100 10500 15100 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 45 99 45 99 43 101 UO only Median 60900 73800 60900 71700 50700 74200 Average 66000 111000 67200 105000 62000 105000 Stdev 35600 165000 37000 157000 35000 151000 p (t-test) 0.042 0.091 0.059 Min 15100 23300 15100 23300 15100 23300 Max 181000 1230000 181000 1230000 181000 1230000 n (Patient) 60 65 52 73 47 79 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.67 0.64 0.70 0.67 0.61 0.70 0.67 0.67 SE 0.047 0.046 0.049 0.048 0.046 0.050 0.048 0.047 0.048 p Value 1.4E−4 3.7E−4 0.0035 3.4E−5 3.7E−4 0.022 2.4E−5 3.6E−4 4.1E−4 nCohort Recovered 38 45 60 33 45 52 31 43 47 nCohort Non-recovered 106 99 65 111 99 73 113 101 79 Cutoff Quartile 2 41800 41800 45200 41800 41800 45200 41800 41800 44800 Sensitivity 82% 83% 83% 81% 83% 79% 81% 83% 81% Specificity 45% 42% 33% 45% 42% 31% 48% 44% 36% Cutoff Quartile 3 65200 65200 68200 65200 65200 68200 65200 65200 68000 Sensitivity 57% 58% 58% 57% 58% 56% 57% 57% 58% Specificity 68% 67% 58% 73% 67% 58% 74% 67% 64% Cutoff Quartile 4 92300 92300 95500 92300 92300 95500 92300 92300 95100 Sensitivity 31% 30% 35% 30% 30% 32% 29% 30% 32% Specificity 92% 87% 85% 91% 87% 83% 90% 86% 85% OR Quartile 2 3.71 3.52 2.45 3.57 3.52 1.72 4.11 3.91 2.42 p Value 0.0015 0.0018 0.037 0.0028 0.0018 0.19 0.0011 7.9E−4 0.035 Lower limit of 95% CI 1.65 1.60 1.06 1.55 1.60 0.758 1.76 1.76 1.07 Upper limit of 95% CI 8.33 7.76 5.70 8.22 7.76 3.89 9.60 8.67 5.48 OR Quartile 3 2.83 2.71 1.97 3.50 2.71 1.75 3.76 2.79 2.46 p Value 0.0094 0.0079 0.062 0.0040 0.0079 0.13 0.0034 0.0072 0.018 Lower limit of 95% CI 1.29 1.30 0.967 1.49 1.30 0.852 1.55 1.32 1.17 Upper limit of 95% CI 6.19 5.67 4.02 8.21 5.67 3.58 9.11 5.91 5.18 OR Quartile 4 5.27 2.83 3.10 4.23 2.83 2.20 3.85 2.61 2.65 p Value 0.0091 0.034 0.011 0.024 0.034 0.077 0.036 0.051 0.041 Lower limit of 95% CI 1.51 1.08 1.30 1.21 1.08 0.919 1.09 0.995 1.04 Upper limit of 95% CI 18.4 7.38 7.42 14.8 7.38 5.26 13.5 6.82 6.72

TABLE 6.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 48400 73800 48200 73900 48200 73900 Average 68600 101000 60800 109000 60800 109000 Stdev 80100 145000 39500 160000 39500 160000 p (t-test) 0.087 0.0096 0.0096 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 265000 1230000 265000 1230000 n (Patient) 81 69 80 70 80 70 sCr only Median 45400 70900 45400 70900 45400 70900 Average 68200 95700 67500 95600 67500 95600 Stdev 87400 133000 88200 131000 88200 131000 p (t-test) 0.15 0.14 0.14 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 67 83 65 85 65 85 UO only Median 50700 72700 50300 77200 49800 81200 Average 70300 107000 63200 118000 62000 118000 Stdev 76000 162000 40800 177000 40100 174000 p (t-test) 0.059 0.0048 0.0037 Min 10500 21500 10500 21500 10500 21500 Max 689000 1230000 265000 1230000 265000 1230000 n (Patient) 95 54 93 56 91 58 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.69 0.67 0.69 0.70 0.69 0.69 0.70 0.71 SE 0.044 0.043 0.047 0.044 0.042 0.046 0.044 0.042 0.045 p Value 8.3E−5 8.4E−6 2.7E−4 1.8E−5 3.3E−6 3.0E−5 1.8E−5 3.3E−6 3.6E−6 nCohort Recovered 81 67 95 80 65 93 80 65 91 nCohort Non-recovered 69 83 54 70 85 56 70 85 58 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 86% 86% 89% 86% 86% 89% 86% 86% 90% Specificity 35% 39% 33% 35% 40% 33% 35% 40% 34% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 62% 63% 65% 63% 62% 66% 63% 62% 67% Specificity 60% 66% 58% 61% 66% 59% 61% 66% 60% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 36% 34% 37% 37% 34% 39% 37% 34% 40% Specificity 84% 85% 81% 85% 86% 83% 85% 86% 84% OR Quartile 2 3.12 3.75 3.88 3.23 4.06 4.17 3.23 4.06 4.48 p Value 0.0061 9.6E−4 0.0052 0.0046 4.9E−4 0.0032 0.0046 4.9E−4 0.0020 Lower limit of 95% CI 1.38 1.71 1.50 1.43 1.85 1.61 1.43 1.85 1.73 Upper limit of 95% CI 7.02 8.22 10.0 7.28 8.91 10.8 7.28 8.91 11.6 OR Quartile 3 2.53 3.21 2.53 2.67 3.24 2.82 2.67 3.24 3.14 p Value 0.0058 6.8E−4 0.0084 0.0035 6.5E−4 0.0033 0.0035 6.5E−4 0.0012 Lower limit of 95% CI 1.31 1.64 1.27 1.38 1.65 1.41 1.38 1.65 1.57 Upper limit of 95% CI 4.90 6.29 5.06 5.18 6.36 5.62 5.18 6.36 6.26 OR Quartile 4 2.97 2.90 2.52 3.35 3.22 3.11 3.35 3.22 3.33 p Value 0.0056 0.010 0.016 0.0025 0.0060 0.0034 0.0025 0.0060 0.0020 Lower limit of 95% CI 1.38 1.29 1.18 1.53 1.40 1.46 1.53 1.40 1.55 Upper limit of 95% CI 6.42 6.53 5.35 7.32 7.42 6.66 7.32 7.42 7.15

Example 7. Use of Follistatin-Related Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Follistatin-related protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 7.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 37600 70200 37100 70200 36900 70000 Average 50400 92400 48900 92100 49000 91700 Stdev 45400 126000 45900 125000 46700 125000 p (t-test) 0.067 0.066 0.073 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 32 118 30 120 29 121 sCr only Median 40400 70500 40400 70500 39100 70200 Average 67200 89500 67200 89500 67800 89100 Stdev 108000 118000 108000 118000 110000 117000 p (t-test) 0.29 0.29 0.32 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 41 109 41 109 40 110 UO only Median 48200 81800 48200 81800 47900 81200 Average 60200 108000 59900 107000 59800 107000 Stdev 41300 156000 41500 154000 41800 154000 p (t-test) 0.011 0.012 0.013 Min 10500 21500 10500 21500 10500 21500 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 76 73 74 75 73 76 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.76 0.74 0.70 0.78 0.74 0.71 0.78 0.74 0.71 SE 0.042 0.042 0.043 0.041 0.042 0.042 0.041 0.042 0.042 p Value 3.4E−10 8.9E−9 2.0E−6 5.3E−12 8.9E−9 1.2E−6 1.0E−11 1.8E−8 1.0E−6 nCohort Recovered 32 41 76 30 41 74 29 40 73 nCohort Non-recovered 118 109 73 120 109 75 121 110 76 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 83% 84% 86% 83% 84% 87% 83% 85% 87% Specificity 56% 51% 36% 60% 51% 36% 62% 52% 37% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 59% 61% 67% 59% 61% 67% 59% 60% 67% Specificity 84% 78% 66% 87% 78% 66% 86% 78% 67% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 30% 30% 37% 30% 30% 37% 30% 30% 37% Specificity 91% 88% 86% 93% 88% 86% 93% 88% 86% OR Quartile 2 6.30 5.68 3.47 7.50 5.68 3.73 8.26 6.05 3.87 p Value 2.1E−5 2.2E−5 0.0028 6.2E−6 2.2E−5 0.0016 3.3E−6 1.3E−5 0.0012 Lower limit of 95% CI 2.70 2.55 1.54 3.13 2.55 1.65 3.39 2.70 1.71 Upper limit of 95% CI 14.7 12.7 7.85 18.0 12.7 8.45 20.1 13.6 8.77 OR Quartile 3 7.88 5.46 3.93 9.42 5.46 3.92 8.88 5.17 4.16 p Value 7.6E−5 6.6E−5 8.2E−5 7.9E−5 6.6E−5 8.3E−5 1.3E−4 1.1E−4 4.3E−5 Lower limit of 95% CI 2.83 2.37 1.99 3.09 2.37 1.99 2.91 2.24 2.10 Upper limit of 95% CI 21.9 12.6 7.75 28.7 12.6 7.74 27.1 11.9 8.25 OR Quartile 4 4.08 3.13 3.47 6.00 3.13 3.81 5.72 3.00 3.68 p Value 0.028 0.029 0.0022 0.018 0.029 0.0013 0.022 0.035 0.0017 Lower limit of 95% CI 1.16 1.13 1.56 1.36 1.13 1.69 1.29 1.08 1.63 Upper limit of 95% CI 14.3 8.68 7.69 26.5 8.68 8.61 25.3 8.34 8.29

TABLE 7.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 40400 71700 38700 71700 38700 71700 Average 49900 97800 48800 97400 48800 97400 Stdev 39700 133000 39800 132000 39800 132000 p (t-test) 0.019 0.019 0.019 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 45 105 43 107 43 107 sCr only Median 41800 71900 41800 71700 41800 71700 Average 63300 93500 63700 93000 63700 93000 Stdev 98400 122000 99400 121000 99400 121000 p (t-test) 0.13 0.14 0.14 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 50 100 49 101 49 101 UO only Median 49100 81200 48400 81200 48400 81200 Average 60500 113000 59900 112000 59900 112000 Stdev 39600 164000 39800 161000 39800 161000 p (t-test) 0.0069 0.0071 0.0071 Min 10500 21500 10500 21500 10500 21500 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 79 66 77 68 77 68 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.78 0.76 0.71 0.79 0.76 0.72 0.79 0.76 0.72 SE 0.038 0.039 0.043 0.037 0.039 0.043 0.037 0.039 0.043 p Value 1.6E−13 8.2E−12 8.2E−7 5.6E−15 2.1E−11 2.2E−7 5.6E−15 2.1E−11 2.2E−7 nCohort Recovered 45 50 79 43 49 77 43 49 77 nCohort Non-recovered 105 100 66 107 101 68 107 101 68 Cutoff Quartile 2 40800 40800 41800 40800 40800 41800 40800 40800 41800 Sensitivity 86% 86% 89% 86% 86% 90% 86% 86% 90% Specificity 51% 48% 37% 53% 49% 38% 53% 49% 38% Cutoff Quartile 3 63600 63600 64300 63600 63600 64300 63600 63600 64300 Sensitivity 64% 65% 67% 64% 64% 68% 64% 64% 68% Specificity 82% 80% 63% 84% 80% 65% 84% 80% 65% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 33% 33% 38% 34% 33% 38% 34% 33% 38% Specificity 93% 90% 85% 95% 90% 86% 95% 90% 86% OR Quartile 2 6.27 5.67 4.89 7.05 5.97 5.26 7.05 5.97 5.26 p Value 6.9E−6 1.7E−5 6.1E−4 2.3E−6 1.1E−5 3.4E−4 2.3E−6 1.1E−5 3.4E−4 Lower limit of 95% CI 2.82 2.57 1.97 3.14 2.69 2.12 3.14 2.69 2.12 Upper limit of 95% CI 14.0 12.5 12.1 15.9 13.2 13.1 15.9 13.2 13.1 OR Quartile 3 8.15 7.43 3.45 8.97 7.04 3.87 8.97 7.04 3.87 p Value 1.8E−6 1.1E−6 4.1E−4 1.8E−6 2.0E−6 1.2E−4 1.8E−6 2.0E−6 1.2E−4 Lower limit of 95% CI 3.44 3.32 1.74 3.64 3.15 1.94 3.64 3.15 1.94 Upper limit of 95% CI 19.3 16.6 6.85 22.1 15.8 7.73 22.1 15.8 7.73 OR Quartile 4 7.00 4.43 3.40 10.4 4.27 3.71 10.4 4.27 3.71 p Value 0.0021 0.0040 0.0024 0.0019 0.0050 0.0014 0.0019 0.0050 0.0014 Lower limit of 95% CI 2.03 1.61 1.54 2.38 1.55 1.66 2.38 1.55 1.66 Upper limit of 95% CI 24.2 12.2 7.50 45.4 11.8 8.30 45.4 11.8 8.30

TABLE 7.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 43500 72000 43500 72000 43600 71900 Average 55600 102000 55600 102000 56000 101000 Stdev 40500 142000 40500 142000 40700 141000 p (t-test) 0.017 0.017 0.020 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 58 91 58 91 57 92 sCr only Median 43900 71700 43900 71700 43900 71700 Average 67700 95600 67700 95600 67700 95600 Stdev 90500 130000 90500 130000 90500 130000 p (t-test) 0.15 0.15 0.15 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 61 87 61 87 61 87 UO only Median 52000 81800 52000 81800 52000 81800 Average 62100 116000 62100 116000 61900 114000 Stdev 33300 169000 33300 169000 33100 167000 p (t-test) 0.0094 0.0094 0.011 Min 15100 24400 15100 24400 15100 24400 Max 181000 1230000 181000 1230000 181000 1230000 n (Patient) 72 61 72 61 70 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.71 0.70 0.74 0.71 0.70 0.73 0.71 0.69 SE 0.040 0.042 0.046 0.040 0.042 0.046 0.041 0.042 0.046 p Value 3.8E−9 1.2E−6 1.8E−5 3.8E−9 1.2E−6 1.8E−5 2.7E−8 1.2E−6 2.8E−5 nCohort Recovered 58 61 72 58 61 72 57 61 70 nCohort Non-recovered 91 87 61 91 87 61 92 87 63 Cutoff Quartile 2 41800 41500 43900 41800 41500 43900 41800 41500 43900 Sensitivity 87% 86% 87% 87% 86% 87% 86% 86% 86% Specificity 43% 41% 35% 43% 41% 35% 42% 41% 34% Cutoff Quartile 3 63900 64100 66600 63900 64100 66600 63900 64100 66600 Sensitivity 64% 62% 66% 64% 62% 66% 63% 62% 65% Specificity 71% 67% 62% 71% 67% 62% 70% 67% 63% Cutoff Quartile 4 91300 91400 93500 91300 91400 93500 91300 91400 93500 Sensitivity 37% 34% 38% 37% 34% 38% 37% 34% 38% Specificity 93% 89% 85% 93% 89% 85% 93% 89% 86% OR Quartile 2 4.99 4.34 3.52 4.99 4.34 3.52 4.42 4.34 3.13 p Value 8.1E−5 2.9E−4 0.0054 8.1E−5 2.9E−4 0.0054 2.2E−4 2.9E−4 0.0094 Lower limit of 95% CI 2.24 1.96 1.45 2.24 1.96 1.45 2.01 1.96 1.32 Upper limit of 95% CI 11.1 9.61 8.56 11.1 9.61 8.56 9.72 9.61 7.41 OR Quartile 3 4.24 3.35 3.17 4.24 3.35 3.17 4.01 3.35 3.15 p Value 6.5E−5 5.6E−4 0.0015 6.5E−5 5.6E−4 0.0015 1.2E−4 5.6E−4 0.0015 Lower limit of 95% CI 2.09 1.69 1.56 2.09 1.69 1.56 1.98 1.69 1.55 Upper limit of 95% CI 8.61 6.67 6.47 8.61 6.67 6.47 8.15 6.67 6.41 OR Quartile 4 8.05 4.06 3.36 8.05 4.06 3.36 7.77 4.06 3.69 p Value 2.0E−4 0.0024 0.0040 2.0E−4 0.0024 0.0040 2.6E−4 0.0024 0.0023 Lower limit of 95% CI 2.68 1.65 1.47 2.68 1.65 1.47 2.58 1.65 1.59 Upper limit of 95% CI 24.2 10.0 7.66 24.2 10.0 7.66 23.4 10.0 8.56

TABLE 7.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 44700 80700 44300 80900 44700 80700 Average 67200 100000 57100 107000 56400 106000 Stdev 88100 135000 40700 148000 40000 146000 p (t-test) 0.090 0.011 0.013 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 265000 1230000 265000 1230000 n (Patient) 65 79 62 82 59 85 sCr only Median 45400 74000 45400 74000 45600 73900 Average 68500 100000 68500 100000 69000 99300 Stdev 87100 137000 87100 137000 87600 136000 p (t-test) 0.11 0.11 0.12 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 67 77 67 77 66 78 UO only Median 60900 80700 55400 81200 55200 81200 Average 74300 108000 65100 115000 63700 115000 Stdev 82100 160000 34900 171000 33900 168000 p (t-test) 0.13 0.022 0.019 Min 15100 24400 15100 24400 15100 24400 Max 689000 1230000 181000 1230000 181000 1230000 n (Patient) 70 55 65 60 64 62 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.71 0.66 0.74 0.71 0.67 0.74 0.70 0.68 SE 0.041 0.042 0.050 0.040 0.042 0.048 0.040 0.043 0.048 p Value 1.8E−8 3.9E−7 0.0015 1.2E−9 3.9E−7 5.0E−4 2.3E−9 1.9E−6 2.1E−4 nCohort Recovered 65 67 70 62 67 65 59 66 64 nCohort Non-recovered 79 77 55 82 77 60 85 78 62 Cutoff Quartile 2 41800 41800 45200 41800 41800 45200 41800 41800 44800 Sensitivity 89% 88% 85% 88% 88% 85% 87% 87% 84% Specificity 42% 40% 33% 42% 40% 34% 42% 39% 34% Cutoff Quartile 3 65200 65200 68200 65200 65200 68200 65200 65200 68000 Sensitivity 65% 64% 62% 65% 64% 62% 64% 63% 61% Specificity 68% 66% 59% 69% 66% 60% 69% 65% 61% Cutoff Quartile 4 92300 92300 95500 92300 92300 95500 92300 92300 95100 Sensitivity 37% 35% 38% 38% 35% 38% 38% 35% 39% Specificity 89% 87% 84% 92% 87% 86% 93% 86% 88% OR Quartile 2 5.53 5.10 2.88 5.20 5.10 2.90 4.95 4.42 2.72 p Value 8.3E−5 1.7E−4 0.022 1.0E−4 1.7E−4 0.017 1.3E−4 4.3E−4 0.021 Lower limit of 95% CI 2.36 2.18 1.17 2.26 2.18 1.21 2.18 1.93 1.16 Upper limit of 95% CI 12.9 11.9 7.07 11.9 11.9 6.96 11.2 10.1 6.38 OR Quartile 3 3.82 3.35 2.29 4.14 3.35 2.41 3.97 3.16 2.47 p Value 1.6E−4 5.5E−4 0.025 7.8E−5 5.5E−4 0.016 1.4E−4 9.7E−4 0.013 Lower limit of 95% CI 1.90 1.69 1.11 2.04 1.69 1.18 1.95 1.59 1.21 Upper limit of 95% CI 7.65 6.64 4.71 8.37 6.64 4.95 8.06 6.26 5.06 OR Quartile 4 4.81 3.48 3.31 6.93 3.48 3.87 8.30 3.35 4.42 p Value 7.0E−4 0.0038 0.0053 1.9E−4 0.0038 0.0025 1.8E−4 0.0049 0.0012 Lower limit of 95% CI 1.94 1.50 1.43 2.51 1.50 1.61 2.75 1.44 1.80 Upper limit of 95% CI 11.9 8.09 7.70 19.2 8.09 9.28 25.1 7.80 10.9

TABLE 7.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 48400 74000 48200 77400 47900 80700 Average 68400 105000 68000 105000 60800 115000 Stdev 77400 153000 77700 151000 39700 167000 p (t-test) 0.054 0.050 0.0044 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 265000 1230000 n (Patient) 89 61 88 62 87 63 sCr only Median 47100 74000 47100 74000 47100 74000 Average 67300 101000 67300 101000 67300 101000 Stdev 81300 142000 81300 142000 81300 142000 p (t-test) 0.070 0.070 0.070 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 79 71 79 71 79 71 UO only Median 50500 73800 50500 73800 50100 80700 Average 70000 110000 70000 110000 63100 121000 Stdev 75100 166000 75100 166000 40800 181000 p (t-test) 0.044 0.044 0.0031 Min 10500 33600 10500 33600 10500 33600 Max 689000 1230000 689000 1230000 265000 1230000 n (Patient) 98 51 98 51 96 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.71 0.69 0.70 0.71 0.69 0.71 0.71 0.71 SE 0.045 0.043 0.048 0.044 0.043 0.048 0.044 0.043 0.046 p Value 1.8E−5 1.4E−6 9.0E−5 6.8E−6 1.4E−6 9.0E−5 1.1E−6 1.4E−6 8.1E−6 nCohort Recovered 89 79 98 88 79 98 87 79 96 nCohort Non-recovered 61 71 51 62 71 51 63 71 53 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 89% 87% 90% 89% 87% 90% 89% 87% 91% Specificity 35% 37% 33% 35% 37% 33% 36% 37% 33% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 66% 68% 67% 66% 68% 67% 67% 68% 68% Specificity 61% 66% 58% 61% 66% 58% 62% 66% 59% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 38% 37% 37% 39% 37% 37% 40% 37% 40% Specificity 83% 85% 81% 84% 85% 81% 85% 85% 82% OR Quartile 2 4.12 4.00 4.46 4.27 4.00 4.46 4.43 4.00 4.80 p Value 0.0020 0.0012 0.0039 0.0016 0.0012 0.0039 0.0012 0.0012 0.0024 Lower limit of 95% CI 1.68 1.73 1.62 1.74 1.73 1.62 1.80 1.73 1.74 Upper limit of 95% CI 10.1 9.21 12.3 10.5 9.21 12.3 10.9 9.21 13.2 OR Quartile 3 2.94 4.02 2.78 3.10 4.02 2.78 3.27 4.02 3.10 p Value 0.0018 6.2E−5 0.0046 0.0011 6.2E−5 0.0046 6.3E−4 6.2E−5 0.0017 Lower limit of 95% CI 1.49 2.04 1.37 1.57 2.04 1.37 1.66 2.04 1.53 Upper limit of 95% CI 5.79 7.94 5.64 6.11 7.94 5.64 6.46 7.94 6.27 OR Quartile 4 2.99 3.23 2.47 3.34 3.23 2.47 3.74 3.23 3.05 p Value 0.0047 0.0033 0.019 0.0021 0.0033 0.019 8.5E−4 0.0033 0.0040 Lower limit of 95% CI 1.40 1.48 1.16 1.55 1.48 1.16 1.72 1.48 1.43 Upper limit of 95% CI 6.38 7.05 5.26 7.18 7.05 5.26 8.14 7.05 6.52

Example 8. Use of Basigin for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Basigin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 8.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 4230 11600 4090 11500 4090 11500 Average 4730 16600 4430 16500 4430 16500 Stdev 1860 14100 1730 14100 1730 14100 p (t-test) 0.013 0.017 0.017 Min 1970 2640 1970 2640 1970 2640 Max 7150 72100 7010 72100 7010 72100 n (Patient) 9 141 8 142 8 142 sCr only Median 4670 12000 4930 11800 4930 11800 Average 9050 16800 9330 16700 9330 16700 Stdev 11700 14000 12000 14000 12000 14000 p (t-test) 0.027 0.040 0.040 Min 1970 3040 1970 3040 1970 3040 Max 42400 72100 42400 72100 42400 72100 n (Patient) 18 132 17 133 17 133 UO only Median 7550 14000 7740 13900 7550 13900 Average 10700 19700 10700 19400 10500 19300 Stdev 10100 15200 10200 15100 10000 15000 p (t-test) 7.3E−5 1.4E−4 1.3E−4 Min 1970 2640 1970 2640 1970 2640 Max 60400 72100 60400 72100 60400 72100 n (Patient) 63 86 60 89 57 92 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.89 0.79 0.73 0.91 0.78 0.72 0.91 0.78 0.73 SE 0.038 0.046 0.041 0.034 0.049 0.041 0.034 0.049 0.041 p Value 0 2.5E−10 1.7E−8 0 1.3E−8 5.6E−8 0 1.3E−8 3.9E−8 nCohort Recovered 9 18 63 8 17 60 8 17 57 nCohort Non-recovered 141 132 86 142 133 89 142 133 92 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 78% 80% 84% 78% 80% 83% 78% 80% 83% Specificity 78% 67% 37% 88% 65% 37% 88% 65% 37% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 53% 55% 70% 53% 54% 69% 53% 54% 67% Specificity 100%  83% 76% 100%  82% 77% 100%  82% 77% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 27% 27% 35% 27% 27% 35% 27% 27% 35% Specificity 100%  89% 87% 100%  88% 88% 100%  88% 89% OR Quartile 2 12.4 8.15 2.96 25.1 7.20 2.86 25.1 7.20 2.77 p Value 0.0023 1.2E−4 0.0057 0.0031 3.4E−4 0.0071 0.0031 3.4E−4 0.0087 Lower limit of 95% CI 2.45 2.80 1.37 2.97 2.44 1.33 2.97 2.44 1.29 Upper limit of 95% CI 62.8 23.8 6.38 212 21.2 6.13 212 21.2 5.94 OR Quartile 3 21.6 6.00 7.38 19.0 5.51 7.16 19.0 5.51 6.99 p Value 0.035 0.0063 1.2E−7 0.044 0.0097 2.4E−7 0.044 0.0097 4.7E−7 Lower limit of 95% CI 1.23 1.66 3.52 1.08 1.51 3.39 1.08 1.51 3.28 Upper limit of 95% CI 378 21.7 15.5 336 20.1 15.1 336 20.1 14.9 OR Quartile 4 7.07 3.00 3.68 6.26 2.78 4.05 6.26 2.78 4.53 p Value 0.18 0.16 0.0031 0.21 0.19 0.0024 0.21 0.19 0.0018 Lower limit of 95% CI 0.402 0.657 1.55 0.353 0.606 1.64 0.353 0.606 1.76 Upper limit of 95% CI 124 13.7 8.74 111 12.8 9.96 111 12.8 11.7

TABLE 8.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 4390 12400 4260 12300 4230 12300 Average 4740 17700 4600 17400 4620 17300 Stdev 1650 14200 1680 14200 1730 14200 p (t-test) 5.7E−5 2.0E−4 3.3E−4 Min 1970 3040 1970 3040 1970 3040 Max 7920 72100 7920 72100 7920 72100 n (Patient) 21 129 18 132 17 133 sCr only Median 4660 12800 4850 12600 4930 12400 Average 7670 17700 7930 17500 8070 17400 Stdev 9500 14200 9820 14100 10000 14100 p (t-test) 4.7E−4 0.0012 0.0020 Min 1970 3040 1970 3040 1970 3040 Max 42400 72100 42400 72100 42400 72100 n (Patient) 28 122 26 124 25 125 UO only Median 7920 14300 8010 13900 7740 13900 Average 11600 20500 11800 19800 11500 19800 Stdev 10500 15700 10800 15500 10600 15400 p (t-test) 1.0E−4 5.1E−4 3.1E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 71 74 66 79 64 81 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.93 0.85 0.73 0.93 0.84 0.70 0.93 0.83 0.71 SE 0.022 0.033 0.042 0.022 0.035 0.043 0.023 0.037 0.042 p Value 0 0 6.7E−8 0 0 1.6E−6 0 0 3.3E−7 nCohort Recovered 21 28 71 18 26 66 17 25 64 nCohort Non-recovered 129 122 74 132 124 79 133 125 81 Cutoff Quartile 2 6820 6820 6850 6820 6820 6850 6820 6820 6850 Sensitivity 84% 85% 88% 83% 84% 86% 82% 83% 86% Specificity 81% 71% 38% 83% 69% 38% 82% 68% 39% Cutoff Quartile 3 11000 11000 11200 11000 11000 11200 11000 11000 11200 Sensitivity 58% 59% 73% 57% 58% 70% 56% 58% 69% Specificity 100%  89% 72% 100%  88% 71% 100%  88% 72% Cutoff Quartile 4 19900 19900 21500 19900 19900 21500 19900 19900 21500 Sensitivity 29% 30% 35% 29% 29% 34% 29% 29% 35% Specificity 100%  93% 85% 100%  92% 85% 100%  92% 86% OR Quartile 2 21.9 14.4 4.43 23.7 11.7 3.77 21.2 10.5 4.08 p Value 3.4E−7 5.1E−8 5.6E−4 2.5E−6 5.2E−7 0.0013 6.1E−6 1.6E−6 6.7E−4 Lower limit of 95% CI 6.68 5.53 1.90 6.34 4.48 1.68 5.65 4.02 1.81 Upper limit of 95% CI 71.5 37.7 10.3 88.6 30.6 8.46 79.6 27.5 9.17 OR Quartile 3 59.6 12.0 6.88 48.6 10.6 5.67 45.2 9.96 5.72 p Value 0.0046 9.9E−5 2.1E−7 0.0072 2.2E−4 2.1E−6 0.0084 3.4E−4 2.1E−6 Lower limit of 95% CI 3.53 3.44 3.32 2.87 3.03 2.77 2.66 2.83 2.78 Upper limit of 95% CI 1000 41.9 14.3 823 37.2 11.6 767 35.0 11.8 OR Quartile 4 18.1 5.44 2.95 15.1 4.91 2.91 14.1 4.65 3.23 p Value 0.045 0.026 0.0080 0.061 0.037 0.011 0.067 0.044 0.0063 Lower limit of 95% CI 1.07 1.23 1.33 0.886 1.10 1.28 0.828 1.04 1.39 Upper limit of 95% CI 306 24.1 6.58 256 21.9 6.59 241 20.8 7.48

TABLE 8.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 6250 12900 6250 12700 5550 12600 Average 8350 18000 8490 17800 7790 17700 Stdev 7550 14600 7770 14500 7410 14400 p (t-test) 4.1E−4 9.0E−4 6.6E−4 Min 1970 3580 1970 3580 1970 3580 Max 33400 72100 33400 72100 33400 72100 n (Patient) 32 117 30 119 27 122 sCr only Median 7010 13200 7010 13100 7010 13100 Average 10000 18100 10200 18000 10200 18000 Stdev 10000 14500 10100 14500 10100 14500 p (t-test) 0.0019 0.0031 0.0031 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 42400 72100 42400 72100 n (Patient) 38 110 37 111 37 111 UO only Median 9030 13900 9000 13900 9000 13600 Average 13300 20300 13200 20200 13400 19500 Stdev 11300 16000 11300 15900 11700 15600 p (t-test) 0.0045 0.0046 0.016 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 64 69 61 72 55 78 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.79 0.75 0.66 0.78 0.74 0.66 0.81 0.74 0.64 SE 0.039 0.041 0.047 0.041 0.042 0.047 0.039 0.042 0.048 p Value 1.1E−13 9.2E−10 6.0E−4 4.3E−12 1.1E−8 4.2E−4 8.9E−16 1.1E−8 0.0024 nCohort Recovered 32 38 64 30 37 61 27 37 55 nCohort Non-recovered 117 110 69 119 111 72 122 111 78 Cutoff Quartile 2 6850 6960 7010 6850 6960 7010 6850 6960 7010 Sensitivity 83% 83% 84% 82% 82% 85% 83% 82% 82% Specificity 53% 47% 33% 53% 46% 34% 59% 46% 33% Cutoff Quartile 3 11000 11100 11800 11000 11100 11800 11000 11100 11800 Sensitivity 60% 60% 65% 59% 59% 64% 59% 59% 60% Specificity 84% 79% 66% 83% 78% 66% 89% 78% 64% Cutoff Quartile 4 20100 20400 23200 20100 20400 23200 20100 20400 23200 Sensitivity 30% 29% 33% 29% 29% 33% 30% 29% 32% Specificity 91% 87% 83% 90% 86% 84% 93% 86% 84% OR Quartile 2 5.50 4.31 2.58 5.33 3.87 2.91 7.00 3.87 2.22 p Value 7.7E−5 3.8E−4 0.025 1.3E−4 0.0010 0.012 2.3E−5 0.0010 0.052 Lower limit of 95% CI 2.36 1.92 1.12 2.26 1.72 1.27 2.84 1.72 0.992 Upper limit of 95% CI 12.8 9.65 5.90 12.6 8.67 6.69 17.2 8.67 4.99 OR Quartile 3 8.04 5.62 3.58 7.14 5.32 3.37 11.5 5.32 2.65 p Value 6.5E−5 9.6E−5 4.7E−4 1.8E−4 1.7E−4 8.6E−4 1.3E−4 1.7E−4 0.0073 Lower limit of 95% CI 2.89 2.36 1.75 2.56 2.23 1.65 3.29 2.23 1.30 Upper limit of 95% CI 22.4 13.4 7.32 20.0 12.7 6.88 40.3 12.7 5.41 OR Quartile 4 4.13 2.71 2.41 3.75 2.59 2.55 5.23 2.59 2.41 p Value 0.027 0.057 0.036 0.039 0.069 0.028 0.030 0.069 0.044 Lower limit of 95% CI 1.18 0.970 1.06 1.07 0.927 1.10 1.18 0.927 1.02 Upper limit of 95% CI 14.4 7.56 5.47 13.2 7.25 5.89 23.3 7.25 5.69

TABLE 8.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 7010 13600 6300 13400 6300 13100 Average 9170 18700 8670 18400 8830 18200 Stdev 8610 14800 8740 14600 8980 14600 p (t-test) 2.7E−4 3.8E−4 8.7E−4 Min 1970 3580 1970 3580 1970 3580 Max 40700 72100 40700 72100 40700 72100 n (Patient) 38 106 33 111 31 113 sCr only Median 7010 13700 7010 13700 7010 13600 Average 10400 18800 10400 18800 10600 18600 Stdev 10300 14800 10300 14800 10500 14800 p (t-test) 6.9E−4 6.9E−4 0.0015 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 42400 72100 42400 72100 n (Patient) 45 99 45 99 43 101 UO only Median 9380 15600 9380 13700 9000 13900 Average 13300 21200 13700 20100 12400 20200 Stdev 10700 16300 11200 15800 10600 15600 p (t-test) 0.0020 0.014 0.0031 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 60 65 52 73 47 79 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.78 0.76 0.67 0.80 0.76 0.64 0.79 0.75 0.68 SE 0.039 0.040 0.048 0.038 0.040 0.049 0.040 0.041 0.047 p Value 6.5E−13 5.8E−11 4.9E−4 1.1E−15 5.8E−11 0.0055 1.3E−13 1.7E−9 1.5E−4 nCohort Recovered 38 45 60 33 45 52 31 43 47 nCohort Non-recovered 106 99 65 111 99 73 113 101 79 Cutoff Quartile 2 7010 7010 7530 7010 7010 7530 7010 7010 7250 Sensitivity 83% 84% 85% 84% 84% 81% 83% 83% 82% Specificity 47% 44% 35% 55% 44% 33% 55% 44% 38% Cutoff Quartile 3 11100 11100 12400 11100 11100 12400 11100 11100 12300 Sensitivity 62% 64% 63% 61% 64% 59% 60% 62% 59% Specificity 84% 80% 63% 88% 80% 62% 87% 79% 66% Cutoff Quartile 4 21600 21600 23800 21600 21600 23800 21600 21600 23600 Sensitivity 30% 30% 35% 30% 30% 33% 29% 30% 34% Specificity 89% 87% 85% 91% 87% 85% 90% 86% 89% OR Quartile 2 4.40 4.15 2.96 6.20 4.15 2.05 6.01 3.91 2.88 p Value 3.6E−4 4.5E−4 0.013 2.6E−5 4.5E−4 0.088 4.6E−5 7.9E−4 0.012 Lower limit of 95% CI 1.95 1.87 1.26 2.65 1.87 0.900 2.54 1.76 1.26 Upper limit of 95% CI 9.93 9.19 6.98 14.5 9.19 4.66 14.2 8.67 6.57 OR Quartile 3 8.80 7.00 2.95 11.5 7.00 2.29 10.2 6.26 2.85 p Value 8.3E−6 5.3E−6 0.0036 1.7E−5 5.3E−6 0.025 4.5E−5 1.8E−5 0.0064 Lower limit of 95% CI 3.38 3.03 1.43 3.77 3.03 1.11 3.34 2.71 1.34 Upper limit of 95% CI 22.9 16.2 6.11 34.9 16.2 4.75 31.1 14.5 6.04 OR Quartile 4 3.68 2.83 3.10 4.23 2.83 2.69 3.85 2.61 4.36 p Value 0.022 0.034 0.011 0.024 0.034 0.031 0.036 0.051 0.0054 Lower limit of 95% CI 1.20 1.08 1.30 1.21 1.08 1.10 1.09 0.995 1.55 Upper limit of 95% CI 11.2 7.38 7.42 14.8 7.38 6.61 13.5 6.82 12.3

TABLE 8.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 8090 13900 8010 14000 8010 14000 Average 12200 20100 11800 20500 11800 20500 Stdev 11000 15800 10500 15900 10500 15900 p (t-test) 4.2E−4 1.2E−4 1.2E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 81 69 80 70 80 70 sCr only Median 7530 13900 7500 13900 7500 13900 Average 10800 19900 10500 19900 10500 19900 Stdev 9540 15600 9400 15500 9400 15500 p (t-test) 4.5E−5 2.9E−5 2.9E−5 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 42400 72100 42400 72100 n (Patient) 67 83 65 85 65 85 UO only Median 8740 16800 8720 17000 8440 16800 Average 12500 21900 12000 22300 12000 22000 Stdev 10600 17000 10200 16900 10300 16700 p (t-test) 4.8E−5 7.5E−6 1.3E−5 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 95 54 93 56 91 58 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.74 0.71 0.72 0.75 0.73 0.72 0.75 0.73 SE 0.043 0.040 0.046 0.042 0.039 0.044 0.042 0.039 0.044 p Value 2.3E−6 1.7E−9 5.3E−6 3.7E−7 2.0E−10 3.5E−7 3.7E−7 2.0E−10 2.1E−7 nCohort Recovered 81 67 95 80 65 93 80 65 91 nCohort Non-recovered 69 83 54 70 85 56 70 85 58 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 88% 88% 91% 89% 88% 91% 89% 88% 91% Specificity 37% 42% 34% 38% 43% 34% 38% 43% 35% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 68% 67% 72% 69% 67% 73% 69% 67% 74% Specificity 65% 72% 62% 66% 72% 63% 66% 72% 65% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 33% 35% 37% 34% 35% 39% 34% 35% 38% Specificity 81% 87% 81% 82% 88% 83% 82% 88% 82% OR Quartile 2 4.49 5.24 4.98 4.65 5.68 5.35 4.65 5.68 5.75 p Value 6.6E−4 7.5E−5 0.0019 4.9E−4 3.5E−5 0.0012 4.9E−4 3.5E−5 7.1E−4 Lower limit of 95% CI 1.89 2.31 1.81 1.96 2.49 1.94 1.96 2.49 2.09 Upper limit of 95% CI 10.6 11.9 13.7 11.0 12.9 14.7 11.0 12.9 15.8 OR Quartile 3 4.04 5.24 4.26 4.28 5.32 4.74 4.28 5.32 5.29 p Value 6.0E−5 3.8E−6 9.0E−5 3.2E−5 3.6E−6 2.7E−5 3.2E−5 3.6E−6 7.5E−6 Lower limit of 95% CI 2.04 2.60 2.06 2.16 2.62 2.29 2.16 2.62 2.55 Upper limit of 95% CI 8.00 10.6 8.80 8.50 10.8 9.81 8.50 10.8 11.0 OR Quartile 4 2.20 3.46 2.52 2.46 3.89 3.11 2.46 3.89 2.86 p Value 0.040 0.0036 0.016 0.020 0.0021 0.0034 0.020 0.0021 0.0064 Lower limit of 95% CI 1.04 1.50 1.18 1.15 1.64 1.46 1.15 1.64 1.34 Upper limit of 95% CI 4.66 7.97 5.35 5.25 9.22 6.66 5.25 9.22 6.11

Example 9. Use of Basigin for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Basigin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 9.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5080 13000 5080 12800 5060 12700 Average 6710 18300 6620 18200 6660 18100 Stdev 4750 14600 4790 14500 4870 14500 p (t-test) 1.7E−5 3.3E−5 5.2E−5 Min 1970 3040 1970 3040 1970 3040 Max 25700 72100 25700 72100 25700 72100 n (Patient) 32 118 30 120 29 121 sCr only Median 5620 13600 5620 13600 5590 13500 Average 8410 18700 8410 18700 8480 18500 Stdev 8550 14600 8550 14600 8650 14600 p (t-test) 4.0E−5 4.0E−5 6.5E−5 Min 1970 3040 1970 3040 1970 3040 Max 42400 72100 42400 72100 42400 72100 n (Patient) 41 109 41 109 40 110 UO only Median 8160 14100 8160 13900 8090 13900 Average 11600 20400 11700 20100 11700 19900 Stdev 10500 15700 10600 15700 10600 15600 p (t-test) 8.3E−5 1.9E−4 2.6E−4 Min 1970 3170 1970 3170 1970 3170 Max 60400 72100 60400 72100 60400 72100 n (Patient) 76 73 74 75 73 76 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.85 0.82 0.72 0.85 0.82 0.71 0.85 0.81 0.71 SE 0.032 0.034 0.042 0.033 0.034 0.042 0.033 0.035 0.042 p Value 0 0 1.6E−7 0 0 7.2E−7 0 0 9.7E−7 nCohort Recovered 32 41 76 30 41 74 29 40 73 nCohort Non-recovered 118 109 73 120 109 75 121 110 76 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 86% 88% 86% 85% 88% 85% 84% 87% 86% Specificity 66% 61% 36% 67% 61% 35% 66% 60% 36% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 60% 62% 71% 60% 62% 71% 60% 62% 70% Specificity 88% 83% 70% 90% 83% 70% 90% 82% 70% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 31% 32% 36% 31% 32% 35% 31% 32% 34% Specificity 97% 93% 84% 97% 93% 84% 97% 92% 84% OR Quartile 2 11.3 11.5 3.47 11.3 11.5 3.15 10.2 10.3 3.27 p Value 9.6E−8 2.0E−8 0.0028 1.7E−7 2.0E−8 0.0048 5.5E−7 6.5E−8 0.0037 Lower limit of 95% CI 4.65 4.91 1.54 4.56 4.91 1.42 4.11 4.42 1.47 Upper limit of 95% CI 27.7 27.1 7.85 28.1 27.1 7.00 25.3 24.0 7.27 OR Quartile 3 10.6 8.06 5.71 13.5 8.06 5.69 12.7 7.63 5.34 p Value 3.2E−5 5.7E−6 1.3E−6 4.3E−5 5.7E−6 1.3E−6 6.5E−5 1.0E−5 2.7E−6 Lower limit of 95% CI 3.48 3.27 2.82 3.88 3.27 2.82 3.65 3.10 2.65 Upper limit of 95% CI 32.1 19.8 11.5 47.0 19.8 11.5 44.4 18.8 10.8 OR Quartile 4 14.2 5.99 2.95 12.9 5.99 2.74 12.3 5.76 2.64 p Value 0.010 0.0047 0.0066 0.014 0.0047 0.011 0.015 0.0058 0.015 Lower limit of 95% CI 1.86 1.73 1.35 1.70 1.73 1.26 1.62 1.66 1.21 Upper limit of 95% CI 108 20.8 6.44 98.5 20.8 5.98 94.1 20.0 5.76

TABLE 9.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5750 13900 5750 13900 5750 13900 Average 6890 19700 6810 19500 6810 19500 Stdev 4220 14900 4250 14800 4250 14800 p (t-test) 7.3E−8 1.5E−7 1.5E−7 Min 1970 3580 1970 3580 1970 3580 Max 25700 72100 25700 72100 25700 72100 n (Patient) 45 105 43 107 43 107 sCr only Median 5980 14000 6200 13900 6200 13900 Average 8130 19700 8180 19600 8180 19600 Stdev 7800 14700 7870 14700 7870 14700 p (t-test) 6.5E−7 1.2E−6 1.2E−6 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 42400 72100 42400 72100 n (Patient) 50 100 49 101 49 101 UO only Median 8220 15000 8090 14300 8090 14300 Average 12000 21100 12100 20800 12100 20800 Stdev 10700 16000 10800 15900 10800 15900 p (t-test) 7.9E−5 1.5E−4 1.5E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 79 66 77 68 77 68 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.86 0.85 0.72 0.86 0.84 0.72 0.86 0.84 0.72 SE 0.029 0.031 0.043 0.029 0.032 0.043 0.029 0.032 0.043 p Value 0 0 2.7E−7 0 0 3.6E−7 0 0 3.6E−7 nCohort Recovered 45 50 79 43 49 77 43 49 77 nCohort Non-recovered 105 100 66 107 101 68 107 101 68 Cutoff Quartile 2 6820 6820 6850 6820 6820 6850 6820 6820 6850 Sensitivity 90% 91% 89% 89% 90% 90% 89% 90% 90% Specificity 60% 58% 37% 60% 57% 38% 60% 57% 38% Cutoff Quartile 3 11000 11000 11200 11000 11000 11200 11000 11000 11200 Sensitivity 67% 68% 74% 66% 67% 74% 66% 67% 74% Specificity 89% 86% 68% 91% 86% 69% 91% 86% 69% Cutoff Quartile 4 19900 19900 21500 19900 19900 21500 19900 19900 21500 Sensitivity 35% 35% 36% 35% 35% 35% 35% 35% 35% Specificity 98% 94% 84% 98% 94% 83% 98% 94% 83% OR Quartile 2 12.8 14.0 4.89 12.1 12.1 5.26 12.1 12.1 5.26 p Value 7.1E−9 5.4E−9 6.1E−4 1.2E−8 1.5E−8 3.4E−4 1.2E−8 1.5E−8 3.4E−4 Lower limit of 95% CI 5.40 5.76 1.97 5.14 5.11 2.12 5.14 5.11 2.12 Upper limit of 95% CI 30.4 33.9 12.1 28.5 28.8 13.1 28.5 28.8 13.1 OR Quartile 3 16.0 13.1 6.23 19.2 12.4 6.13 19.2 12.4 6.13 p Value 8.5E−8 2.4E−8 8.3E−7 1.5E−7 4.6E−8 8.8E−7 1.5E−7 4.6E−8 8.8E−7 Lower limit of 95% CI 5.80 5.29 3.01 6.37 5.02 2.98 6.37 5.02 2.98 Upper limit of 95% CI 44.1 32.2 12.9 58.0 30.5 12.6 58.0 30.5 12.6 OR Quartile 4 23.9 8.44 2.90 22.2 8.13 2.69 22.2 8.13 2.69 p Value 0.0021 7.3E−4 0.0073 0.0027 9.0E−4 0.013 0.0027 9.0E−4 0.013 Lower limit of 95% CI 3.17 2.45 1.33 2.94 2.36 1.24 2.94 2.36 1.24 Upper limit of 95% CI 181 29.1 6.32 168 28.0 5.84 168 28.0 5.84

TABLE 9.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 7010 14100 7010 14100 7010 14000 Average 9750 19900 9750 19900 9810 19700 Stdev 8860 15200 8860 15200 8930 15100 p (t-test) 8.5E−6 8.5E−6 1.4E−5 Min 1970 3580 1970 3580 1970 3580 Max 40700 72100 40700 72100 40700 72100 n (Patient) 58 91 58 91 57 92 sCr only Median 7150 14100 7150 14100 7150 14100 Average 10900 19700 10900 19700 10900 19700 Stdev 10200 15100 10200 15100 10200 15100 p (t-test) 1.2E−4 1.2E−4 1.2E−4 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 42400 72100 42400 72100 n (Patient) 61 87 61 87 61 87 UO only Median 8970 15600 8970 15600 8970 15600 Average 13000 21600 13000 21600 13000 21400 Stdev 11000 16400 11000 16400 11100 16300 p (t-test) 4.6E−4 4.6E−4 5.9E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 72 61 72 61 70 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.79 0.76 0.70 0.79 0.76 0.70 0.78 0.76 0.70 SE 0.036 0.039 0.046 0.036 0.039 0.046 0.037 0.039 0.046 p Value 2.9E−15 2.6E−11 1.4E−5 2.9E−15 2.6E−11 1.4E−5 2.2E−14 2.6E−11 1.6E−5 nCohort Recovered 58 61 72 58 61 72 57 61 70 nCohort Non-recovered 91 87 61 91 87 61 92 87 63 Cutoff Quartile 2 6850 6960 7010 6850 6960 7010 6850 6960 7010 Sensitivity 89% 89% 89% 89% 89% 89% 88% 89% 87% Specificity 47% 44% 35% 47% 44% 35% 46% 44% 34% Cutoff Quartile 3 11000 11100 11800 11000 11100 11800 11000 11100 11800 Sensitivity 69% 68% 69% 69% 68% 69% 68% 68% 68% Specificity 79% 75% 65% 79% 75% 65% 79% 75% 66% Cutoff Quartile 4 20100 20400 23200 20100 20400 23200 20100 20400 23200 Sensitivity 34% 33% 36% 34% 33% 36% 34% 33% 37% Specificity 88% 87% 83% 88% 87% 83% 88% 87% 84% OR Quartile 2 7.05 6.11 4.10 7.05 6.11 4.10 6.18 6.11 3.59 p Value 4.6E−6 1.9E−5 0.0028 4.6E−6 1.9E−5 0.0028 1.3E−5 1.9E−5 0.0050 Lower limit of 95% CI 3.06 2.67 1.63 3.06 2.67 1.63 2.73 2.67 1.47 Upper limit of 95% CI 16.3 14.0 10.3 16.3 14.0 10.3 14.0 14.0 8.74 OR Quartile 3 8.62 6.46 4.16 8.62 6.46 4.16 8.15 6.46 4.12 p Value 5.2E−8 6.8E−7 1.2E−4 5.2E−8 6.8E−7 1.2E−4 1.1E−7 6.8E−7 1.3E−4 Lower limit of 95% CI 3.97 3.09 2.01 3.97 3.09 2.01 3.76 3.09 2.00 Upper limit of 95% CI 18.7 13.5 8.60 18.7 13.5 8.60 17.7 13.5 8.50 OR Quartile 4 3.76 3.31 2.82 3.76 3.31 2.82 3.63 3.31 3.08 p Value 0.0039 0.0068 0.012 0.0039 0.0068 0.012 0.0051 0.0068 0.0073 Lower limit of 95% CI 1.53 1.39 1.25 1.53 1.39 1.25 1.47 1.39 1.35 Upper limit of 95% CI 9.27 7.88 6.34 9.27 7.88 6.34 8.94 7.88 7.02

TABLE 9.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 7530 14500 7520 15000 7530 14500 Average 10800 20600 10200 20700 10000 20400 Stdev 9670 15600 8860 15600 8680 15500 p (t-test) 2.3E−5 5.0E−6 7.1E−6 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 40700 72100 40700 72100 n (Patient) 65 79 62 82 59 85 sCr only Median 7550 14500 7550 14500 7630 14300 Average 11300 20500 11300 20500 11400 20300 Stdev 10100 15600 10100 15600 10200 15600 p (t-test) 6.6E−5 6.6E−5 1.2E−4 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 42400 72100 42400 72100 n (Patient) 67 77 67 77 66 78 UO only Median 9320 16700 9260 16800 9160 16800 Average 13800 21900 13200 21900 13000 21800 Stdev 11100 16800 10700 16500 10600 16400 p (t-test) 0.0016 5.7E−4 4.3E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 70 55 65 60 64 62 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.76 0.75 0.68 0.78 0.75 0.69 0.77 0.74 0.69 SE 0.039 0.040 0.049 0.038 0.040 0.047 0.038 0.041 0.047 p Value 2.2E−11 7.0E−10 3.2E−4 2.7E−13 7.0E−10 5.9E−5 1.3E−12 5.1E−9 3.6E−5 nCohort Recovered 65 67 70 62 67 65 59 66 64 nCohort Non-recovered 79 77 55 82 77 60 85 78 62 Cutoff Quartile 2 7010 7010 7530 7010 7010 7530 7010 7010 7250 Sensitivity 89% 88% 87% 88% 88% 87% 86% 87% 85% Specificity 42% 40% 34% 42% 40% 35% 41% 39% 36% Cutoff Quartile 3 11100 11100 12400 11100 11100 12400 11100 11100 12300 Sensitivity 71% 70% 67% 71% 70% 67% 69% 69% 66% Specificity 75% 73% 63% 77% 73% 65% 78% 73% 66% Cutoff Quartile 4 21600 21600 23800 21600 21600 23800 21600 21600 23600 Sensitivity 34% 34% 36% 35% 34% 37% 35% 33% 37% Specificity 86% 85% 83% 89% 85% 85% 90% 85% 86% OR Quartile 2 5.53 5.10 3.58 5.20 5.10 3.56 4.17 4.42 3.30 p Value 8.3E−5 1.7E−4 0.0075 1.0E−4 1.7E−4 0.0058 4.8E−4 4.3E−4 0.0072 Lower limit of 95% CI 2.36 2.18 1.41 2.26 2.18 1.45 1.87 1.93 1.38 Upper limit of 95% CI 12.9 11.9 9.10 11.9 11.9 8.77 9.30 10.1 7.90 OR Quartile 3 7.46 6.39 3.48 8.29 6.39 3.65 8.03 6.00 3.73 p Value 1.2E−7 5.9E−7 0.0010 5.4E−8 5.9E−7 5.9E−4 1.1E−7 1.2E−6 4.6E−4 Lower limit of 95% CI 3.54 3.09 1.65 3.87 3.09 1.74 3.72 2.91 1.78 Upper limit of 95% CI 15.7 13.2 7.31 17.8 13.2 7.65 17.3 12.4 7.79 OR Quartile 4 3.23 2.91 2.76 4.30 2.91 3.18 4.82 2.80 3.60 p Value 0.0064 0.011 0.016 0.0016 0.011 0.0079 0.0012 0.014 0.0040 Lower limit of 95% CI 1.39 1.28 1.20 1.73 1.28 1.36 1.86 1.23 1.51 Upper limit of 95% CI 7.51 6.61 6.33 10.7 6.61 7.48 12.5 6.37 8.63

TABLE 9.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 8090 16700 8010 16800 7920 16900 Average 12000 21400 11900 21500 11600 21800 Stdev 10600 16300 10600 16100 10200 16200 p (t-test) 3.4E−5 2.4E−5 5.0E−6 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 89 61 88 62 87 63 sCr only Median 7550 14100 7550 14100 7550 14100 Average 11800 20400 11800 20400 11800 20400 Stdev 11100 15400 11100 15400 11100 15400 p (t-test) 1.3E−4 1.3E−4 1.3E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 79 71 79 71 79 71 UO only Median 8730 16900 8730 16900 8580 17200 Average 12400 22600 12400 22600 12000 23000 Stdev 10500 17200 10500 17200 10100 17000 p (t-test) 1.4E−5 1.4E−5 1.9E−6 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 98 51 98 51 96 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.74 0.72 0.74 0.74 0.72 0.75 0.74 0.74 SE 0.043 0.041 0.046 0.042 0.041 0.046 0.042 0.041 0.044 p Value 8.5E−8 7.3E−9 9.3E−7 2.4E−8 7.3E−9 9.3E−7 2.4E−9 7.3E−9 4.5E−8 nCohort Recovered 89 79 98 88 79 98 87 79 96 nCohort 61 71 51 62 71 51 63 71 53 Non-recovered Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 92% 92% 92% 92% 92% 92% 92% 92% 92% Specificity 37% 41% 34% 38% 41% 34% 38% 41% 34% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 72% 72% 75% 73% 72% 75% 73% 72% 75% Specificity 65% 70% 62% 66% 70% 62% 67% 70% 64% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 36% 35% 39% 37% 35% 39% 38% 35% 42% Specificity 82% 84% 82% 83% 84% 82% 84% 84% 83% OR Quartile 2 6.60 7.38 5.97 6.84 7.38 5.97 7.09 7.38 6.42 p Value 2.5E−4 3.7E−5 0.0015 1.9E−4 3.7E−5 0.0015 1.5E−4 3.7E−5 9.5E−4 Lower limit of 95% CI 2.40 2.85 1.98 2.49 2.85 1.98 2.58 2.85 2.13 Upper limit of 95% CI 18.1 19.1 18.0 18.8 19.1 18.0 19.5 19.1 19.3 OR Quartile 3 4.84 5.84 4.82 5.12 5.84 4.82 5.41 5.84 5.36 p Value 1.3E−5 9.3E−7 4.0E−5 6.8E−6 9.3E−7 4.0E−5 3.4E−6 9.3E−7 1.2E−5 Lower limit of 95% CI 2.38 2.89 2.28 2.51 2.89 2.28 2.65 2.89 2.53 Upper limit of 95% CI 9.85 11.8 10.2 10.4 11.8 10.2 11.0 11.8 11.4 OR Quartile 4 2.57 2.76 2.87 2.87 2.76 2.87 3.21 2.76 3.55 p Value 0.014 0.0097 0.0066 0.0064 0.0097 0.0066 0.0028 0.0097 0.0012 Lower limit of 95% CI 1.21 1.28 1.34 1.35 1.28 1.34 1.49 1.28 1.65 Upper limit of 95% CI 5.46 5.95 6.13 6.12 5.95 6.13 6.90 5.95 7.63

Example 10. Use of Cathepsin B for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Cathepsin B is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 10.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.826 3.83 0.678 3.81 0.678 3.81 Average 1.93 12.6 2.01 12.5 2.01 12.5 Stdev 2.20 20.8 2.34 20.7 2.34 20.7 p (t-test) 0.13 0.16 0.16 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 5.56 111 5.56 111 5.56 111 n (Patient) 9 139 8 140 8 140 sCr only Median 0.917 4.84 0.863 4.83 0.863 4.83 Average 1.72 13.7 1.73 13.6 1.73 13.6 Stdev 1.82 21.5 1.86 21.4 1.86 21.4 p (t-test) 0.010 0.012 0.012 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 6.53 111 6.53 111 6.53 111 n (Patient) 22 126 21 127 21 127 UO only Median 3.60 4.28 3.61 3.83 3.60 4.28 Average 10.3 13.3 10.1 13.3 9.39 13.6 Stdev 17.5 22.2 17.5 22.1 17.1 22.1 p (t-test) 0.37 0.34 0.22 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 63 84 60 87 57 90 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.76 0.78 0.54 0.76 0.78 0.54 0.76 0.78 0.55 SE 0.068 0.045 0.048 0.071 0.046 0.048 0.071 0.046 0.048 p Value 1.3E−4 7.3E−10 0.42 2.8E−4 1.1E−9 0.37 2.8E−4 1.1E−9 0.27 nCohort Recovered 9 22 63 8 21 60 8 21 57 nCohort Non-recovered 139 126 84 140 127 87 140 127 90 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 77% 80% 75% 77% 80% 76% 77% 80% 76% Specificity 56% 55% 25% 62% 57% 27% 62% 57% 26% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 52% 56% 52% 51% 56% 52% 51% 56% 52% Specificity 78% 86% 52% 75% 86% 52% 75% 86% 53% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 27% 29% 26% 26% 29% 26% 26% 29% 28% Specificity 100%  100%  76% 100%  100%  77% 100%  100%  79% OR Quartile 2 4.18 4.85 1.02 5.62 5.44 1.14 5.62 5.44 1.10 p Value 0.041 0.0011 0.96 0.023 6.1E−4 0.73 0.023 6.1E−4 0.80 Lower limit of 95% CI 1.06 1.88 0.481 1.27 2.06 0.538 1.27 2.06 0.516 Upper limit of 95% CI 16.5 12.5 2.17 24.8 14.3 2.43 24.8 14.3 2.36 OR Quartile 3 3.76 8.18 1.21 3.18 7.61 1.15 3.18 7.61 1.21 p Value 0.11 0.0012 0.57 0.17 0.0018 0.69 0.17 0.0018 0.57 Lower limit of 95% CI 0.755 2.30 0.629 0.620 2.13 0.593 0.620 2.13 0.625 Upper limit of 95% CI 18.7 29.0 2.33 16.3 27.1 2.21 16.3 27.1 2.36 OR Quartile 4 6.95 18.9 1.14 6.16 17.8 1.18 6.16 17.8 1.44 p Value 0.19 0.042 0.74 0.22 0.046 0.67 0.22 0.046 0.36 Lower limit of 95% CI 0.395 1.11 0.533 0.347 1.05 0.550 0.347 1.05 0.657 Upper limit of 95% CI 122 319 2.42 109 302 2.54 109 302 3.17

TABLE 10.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.35 4.83 2.06 4.73 1.75 4.77 Average 2.15 13.7 2.40 13.3 2.26 13.3 Stdev 1.90 21.6 2.00 21.4 1.95 21.3 p (t-test) 0.011 0.028 0.031 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 6.09 111 6.09 111 6.09 111 n (Patient) 23 125 19 129 18 130 sCr only Median 1.35 5.87 1.35 4.92 1.32 4.94 Average 2.21 14.7 2.27 14.5 2.18 14.4 Stdev 2.04 22.2 2.09 22.1 2.06 22.0 p (t-test) 0.0016 0.0026 0.0029 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 7.07 111 7.07 111 7.07 111 n (Patient) 33 115 31 117 30 118 UO only Median 3.46 4.81 3.61 4.73 3.61 4.73 Average 9.67 14.3 9.12 14.4 9.09 14.3 Stdev 16.6 23.5 16.1 23.3 16.3 23.0 p (t-test) 0.17 0.12 0.13 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 70 73 64 79 62 81 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.74 0.55 0.69 0.73 0.55 0.70 0.74 0.55 SE 0.051 0.044 0.048 0.058 0.046 0.048 0.058 0.045 0.048 p Value 9.9E−6 5.7E−8 0.30 0.0010 4.2E−7 0.32 4.0E−4 1.2E−7 0.30 nCohort Recovered 23 33 70 19 31 64 18 30 62 nCohort Non-recovered 125 115 73 129 117 79 130 118 81 Cutoff Quartile 2 1.13 1.13 1.06 1.13 1.13 1.06 1.13 1.13 1.06 Sensitivity 78% 80% 75% 77% 79% 76% 77% 80% 75% Specificity 39% 42% 26% 37% 42% 27% 39% 43% 26% Cutoff Quartile 3 3.63 3.63 3.80 3.63 3.63 3.80 3.63 3.63 3.80 Sensitivity 55% 58% 53% 53% 57% 52% 54% 58% 52% Specificity 78% 79% 53% 74% 77% 52% 78% 80% 52% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 30% 32% 27% 29% 32% 28% 28% 31% 28% Specificity 100%  100%  77% 100%  100%  78% 100%  100%  79% OR Quartile 2 2.23 2.95 1.06 1.92 2.80 1.14 2.12 3.00 1.06 p Value 0.094 0.010 0.88 0.21 0.017 0.73 0.15 0.011 0.88 Lower limit of 95% CI 0.873 1.29 0.497 0.696 1.20 0.535 0.756 1.28 0.496 Upper limit of 95% CI 5.68 6.74 2.25 5.33 6.50 2.44 5.95 7.01 2.27 OR Quartile 3 4.44 5.18 1.29 3.22 4.59 1.15 4.08 5.44 1.15 p Value 0.0055 4.1E−4 0.45 0.034 0.0011 0.68 0.018 5.9E−4 0.68 Lower limit of 95% CI 1.55 2.08 0.667 1.10 1.83 0.594 1.28 2.07 0.593 Upper limit of 95% CI 12.7 12.9 2.48 9.46 11.5 2.22 13.1 14.3 2.23 OR Quartile 4 19.9 32.0 1.27 15.8 29.3 1.38 14.8 28.1 1.49 p Value 0.038 0.016 0.53 0.056 0.019 0.41 0.062 0.021 0.31 Lower limit of 95% CI 1.18 1.91 0.596 0.931 1.75 0.638 0.872 1.67 0.686 Upper limit of 95% CI 336 537 2.72 269 493 2.98 253 471 3.26

TABLE 10.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.95 6.29 1.72 5.97 2.02 5.42 Average 2.27 14.9 2.18 14.7 2.34 14.3 Stdev 1.88 22.4 1.88 22.2 1.90 22.1 p (t-test) 0.0013 0.0019 0.0041 Min 0.0435 0.126 0.0435 0.126 0.208 0.0435 Max 7.19 111 7.19 111 7.19 111 n (Patient) 34 113 32 115 29 118 sCr only Median 1.50 6.80 1.44 6.67 1.44 6.67 Average 2.33 16.0 2.26 15.9 2.26 15.9 Stdev 2.05 23.0 2.04 22.9 2.04 22.9 p (t-test) 1.9E−4 2.3E−4 2.3E−4 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 7.19 111 7.19 111 7.19 111 n (Patient) 42 104 41 105 41 105 UO only Median 3.62 5.36 3.71 4.85 4.14 4.73 Average 9.06 16.4 9.13 16.0 9.89 14.9 Stdev 15.9 24.9 16.2 24.5 16.9 23.8 p (t-test) 0.046 0.062 0.18 Min 0.0435 0.126 0.0435 0.126 0.156 0.0435 Max 103 111 103 111 103 111 n (Patient) 65 66 62 69 56 75 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.74 0.59 0.73 0.75 0.59 0.70 0.75 0.54 SE 0.045 0.041 0.050 0.045 0.041 0.050 0.049 0.041 0.051 p Value 1.3E−6 3.9E−9 0.079 4.3E−7 1.2E−9 0.084 5.1E−5 1.2E−9 0.43 nCohort Recovered 34 42 65 32 41 62 29 41 56 nCohort Non-recovered 113 104 66 115 105 69 118 105 75 Cutoff Quartile 2 1.13 1.12 1.16 1.13 1.12 1.16 1.13 1.12 1.16 Sensitivity 79% 81% 80% 79% 81% 80% 78% 81% 76% Specificity 38% 40% 31% 41% 41% 31% 38% 41% 27% Cutoff Quartile 3 3.62 3.63 4.38 3.62 3.63 4.38 3.62 3.63 4.38 Sensitivity 58% 61% 55% 58% 61% 54% 57% 61% 51% Specificity 76% 76% 54% 78% 78% 53% 76% 78% 50% Cutoff Quartile 4 13.1 13.3 14.2 13.1 13.3 14.2 13.1 13.3 14.2 Sensitivity 33% 36% 29% 32% 35% 29% 31% 35% 27% Specificity 100%  100%  78% 100%  100%  79% 100%  100%  77% OR Quartile 2 2.30 2.86 1.81 2.59 3.01 1.74 2.16 3.01 1.16 p Value 0.049 0.0089 0.15 0.026 0.0062 0.18 0.081 0.0062 0.72 Lower limit of 95% CI 1.01 1.30 0.811 1.12 1.37 0.782 0.908 1.37 0.524 Upper limit of 95% CI 5.24 6.27 4.05 5.99 6.63 3.85 5.15 6.63 2.56 OR Quartile 3 4.56 4.92 1.40 4.99 5.55 1.32 4.13 5.55 1.03 p Value 6.8E−4 1.2E−4 0.34 5.9E−4 6.0E−5 0.43 0.0027 6.0E−5 0.94 Lower limit of 95% CI 1.90 2.18 0.704 1.99 2.40 0.662 1.64 2.40 0.514 Upper limit of 95% CI 11.0 11.1 2.78 12.5 12.8 2.62 10.4 12.8 2.05 OR Quartile 4 33.8 47.2 1.47 31.1 45.4 1.54 27.1 45.4 1.20 p Value 0.014 0.0073 0.34 0.017 0.0079 0.29 0.022 0.0079 0.65 Lower limit of 95% CI 2.02 2.82 0.664 1.85 2.72 0.689 1.62 2.72 0.538 Upper limit of 95% CI 567 790 3.26 521 760 3.43 456 760 2.69

TABLE 10.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.55 6.50 1.81 6.13 1.81 6.03 Average 4.26 15.4 4.54 14.7 4.60 14.5 Stdev 9.39 22.9 9.94 22.6 10.2 22.4 p (t-test) 0.0033 0.0097 0.014 Min 0.0435 0.126 0.208 0.0435 0.208 0.0435 Max 57.5 111 57.5 111 57.5 111 n (Patient) 41 101 36 106 34 108 sCr only Median 1.81 6.80 1.81 6.80 1.81 6.58 Average 4.15 16.2 4.15 16.2 4.18 16.0 Stdev 8.71 23.5 8.71 23.5 8.88 23.4 p (t-test) 7.8E−4 7.8E−4 0.0012 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 48 94 48 94 46 96 UO only Median 3.61 5.87 3.85 4.81 4.14 4.77 Average 9.26 16.7 9.70 15.5 10.2 14.9 Stdev 16.1 25.3 16.6 24.4 17.0 23.8 p (t-test) 0.056 0.14 0.23 Min 0.0435 0.126 0.156 0.0435 0.156 0.0435 Max 103 111 103 111 103 111 n (Patient) 60 61 52 69 48 74 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.71 0.60 0.69 0.71 0.56 0.69 0.71 0.53 SE 0.044 0.043 0.051 0.047 0.043 0.052 0.048 0.044 0.053 p Value 1.3E−6 8.0E−7 0.043 6.2E−5 8.0E−7 0.26 9.7E−5 1.5E−6 0.58 nCohort Recovered 41 48 60 36 48 52 34 46 48 nCohort Non-recovered 101 94 61 106 94 69 108 96 74 Cutoff Quartile 2 1.12 1.12 1.22 1.12 1.12 1.22 1.12 1.12 1.23 Sensitivity 80% 81% 80% 79% 81% 77% 79% 80% 73% Specificity 39% 38% 30% 39% 38% 27% 38% 37% 23% Cutoff Quartile 3 3.71 3.71 4.38 3.71 3.71 4.38 3.71 3.71 4.56 Sensitivity 60% 63% 56% 58% 63% 52% 58% 62% 51% Specificity 76% 75% 55% 75% 75% 52% 76% 76% 52% Cutoff Quartile 4 13.3 13.3 16.1 13.3 13.3 16.1 13.3 13.3 15.7 Sensitivity 33% 35% 30% 31% 35% 28% 31% 34% 27% Specificity 93% 94% 78% 92% 94% 77% 91% 93% 77% OR Quartile 2 2.59 2.53 1.75 2.43 2.53 1.22 2.29 2.38 0.803 p Value 0.019 0.019 0.19 0.033 0.019 0.64 0.051 0.030 0.61 Lower limit of 95% CI 1.17 1.16 0.756 1.07 1.16 0.532 0.997 1.09 0.344 Upper limit of 95% CI 5.74 5.52 4.05 5.51 5.52 2.80 5.25 5.19 1.87 OR Quartile 3 4.73 5.06 1.54 4.23 5.06 1.18 4.55 5.30 1.15 p Value 1.9E−4 4.2E−5 0.24 8.6E−4 4.2E−5 0.66 7.4E−4 3.8E−5 0.71 Lower limit of 95% CI 2.09 2.33 0.751 1.81 2.33 0.573 1.89 2.40 0.555 Upper limit of 95% CI 10.7 11.0 3.15 9.87 11.0 2.42 11.0 11.7 2.37 OR Quartile 4 6.15 8.11 1.51 4.97 8.11 1.27 4.55 7.51 1.25 p Value 0.0043 9.6E−4 0.32 0.012 9.6E−4 0.58 0.018 0.0015 0.61 Lower limit of 95% CI 1.77 2.34 0.663 1.42 2.34 0.550 1.30 2.16 0.534 Upper limit of 95% CI 21.4 28.1 3.45 17.4 28.1 2.92 15.9 26.0 2.90

TABLE 10.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.42 6.53 2.46 6.52 2.46 6.52 Average 8.34 16.7 8.43 16.5 8.43 16.5 Stdev 15.4 24.8 15.4 24.6 15.4 24.6 p (t-test) 0.012 0.016 0.016 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 85 63 84 64 84 64 sCr only Median 1.95 8.30 2.04 7.97 2.04 7.97 Average 4.59 18.5 4.71 18.0 4.71 18.0 Stdev 8.07 25.2 8.15 25.0 8.15 25.0 p (t-test) 1.9E−5 4.5E−5 4.5E−5 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 70 78 68 80 68 80 UO only Median 3.05 6.20 3.05 5.36 3.05 5.36 Average 8.92 17.6 9.06 17.0 9.16 16.6 Stdev 15.1 26.7 15.2 26.3 15.4 26.0 p (t-test) 0.013 0.021 0.032 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 95 52 93 54 91 56 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.71 0.60 0.63 0.69 0.59 0.63 0.69 0.58 SE 0.047 0.042 0.050 0.047 0.043 0.049 0.047 0.043 0.049 p Value 0.0030 3.3E−7 0.053 0.0063 1.0E−5 0.082 0.0063 1.0E−5 0.093 nCohort Recovered 85 70 95 84 68 93 84 68 91 nCohort Non-recovered 63 78 52 64 80 54 64 80 56 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 83% 83% 77% 81% 81% 76% 81% 81% 77% Specificity 31% 34% 26% 30% 32% 26% 30% 32% 26% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 62% 65% 60% 61% 64% 59% 61% 64% 59% Specificity 59% 67% 55% 58% 66% 55% 58% 66% 55% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 32% 40% 31% 31% 39% 30% 31% 39% 29% Specificity 80% 91% 78% 80% 91% 77% 80% 91% 77% OR Quartile 2 2.08 2.61 1.19 1.84 2.07 1.10 1.84 2.07 1.18 p Value 0.071 0.015 0.67 0.13 0.059 0.82 0.13 0.059 0.67 Lower limit of 95% CI 0.938 1.20 0.540 0.839 0.972 0.504 0.839 0.972 0.545 Upper limit of 95% CI 4.62 5.65 2.62 4.02 4.42 2.39 4.02 4.42 2.57 OR Quartile 3 2.32 3.86 1.79 2.18 3.44 1.77 2.18 3.44 1.75 p Value 0.013 1.1E−4 0.098 0.021 3.6E−4 0.10 0.021 3.6E−4 0.10 Lower limit of 95% CI 1.19 1.95 0.899 1.12 1.75 0.895 1.12 1.75 0.892 Upper limit of 95% CI 4.52 7.64 3.54 4.24 6.78 3.48 4.24 6.78 3.43 OR Quartile 4 1.86 7.04 1.57 1.79 6.54 1.44 1.79 6.54 1.33 p Value 0.11 5.9E−5 0.25 0.13 1.1E−4 0.34 0.13 1.1E−4 0.46 Lower limit of 95% CI 0.878 2.72 0.730 0.846 2.53 0.675 0.846 2.53 0.625 Upper limit of 95% CI 3.94 18.2 3.36 3.79 16.9 3.09 3.79 16.9 2.84

TABLE 10.6 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 62.2 104 58.6 103 58.6 103 Average 72.8 116 74.2 115 74.2 115 Stdev 33.3 66.6 35.3 66.6 35.3 66.6 p (t-test) 0.057 0.084 0.084 Min 41.1 13.2 41.1 13.2 41.1 13.2 Max 134 401 134 401 134 401 n (Patient) 9 141 8 142 8 142 sCr only Median 65.2 105 68.2 104 68.2 104 Average 81.9 118 83.5 117 83.5 117 Stdev 44.3 67.3 45.1 67.2 45.1 67.2 p (t-test) 0.031 0.048 0.048 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 174 401 174 401 174 401 n (Patient) 18 132 17 133 17 133 UO only Median 91.4 115 92.8 114 94.2 112 Average 98.4 125 98.1 124 99.5 122 Stdev 51.1 73.2 50.5 73.0 51.2 72.4 p (t-test) 0.015 0.018 0.038 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 63 86 60 89 57 92 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.66 0.60 0.71 0.65 0.60 0.71 0.65 0.58 SE 0.076 0.062 0.046 0.081 0.064 0.047 0.081 0.064 0.047 p Value 0.0049 0.0075 0.032 0.012 0.018 0.037 0.012 0.018 0.082 nCohort Recovered 9 18 63 8 17 60 8 17 57 nCohort Non-recovered 141 132 86 142 133 89 142 133 92 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 77% 78% 80% 76% 77% 79% 76% 77% 78% Specificity 56% 50% 32% 50% 47% 30% 50% 47% 30% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 52% 52% 57% 51% 52% 56% 51% 52% 54% Specificity 78% 67% 59% 75% 65% 58% 75% 65% 56% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 27% 27% 30% 27% 27% 30% 27% 27% 29% Specificity 100%  89% 81% 100%  88% 82% 100%  88% 81% OR Quartile 2 4.09 3.55 1.89 3.18 3.05 1.58 3.18 3.05 1.53 p Value 0.044 0.014 0.097 0.12 0.035 0.23 0.12 0.035 0.27 Lower limit of 95% CI 1.04 1.29 0.891 0.754 1.08 0.746 0.754 1.08 0.720 Upper limit of 95% CI 16.1 9.77 4.00 13.4 8.60 3.34 13.4 8.60 3.25 OR Quartile 3 3.76 2.19 1.88 3.17 1.98 1.79 3.17 1.98 1.52 p Value 0.11 0.14 0.059 0.17 0.20 0.083 0.17 0.20 0.21 Lower limit of 95% CI 0.754 0.776 0.975 0.619 0.691 0.925 0.619 0.691 0.784 Upper limit of 95% CI 18.7 6.18 3.64 16.3 5.66 3.48 16.3 5.66 2.96 OR Quartile 4 7.07 3.00 1.84 6.26 2.78 1.94 6.26 2.78 1.74 p Value 0.18 0.16 0.12 0.21 0.19 0.10 0.21 0.19 0.17 Lower limit of 95% CI 0.402 0.657 0.845 0.353 0.606 0.876 0.353 0.606 0.783 Upper limit of 95% CI 124 13.7 4.01 111 12.8 4.30 111 12.8 3.85

TABLE 10.7 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 54.6 111 60.9 105 67.1 104 Average 73.4 120 77.6 118 79.0 118 Stdev 42.2 66.8 44.0 67.0 45.0 66.9 p (t-test) 0.0025 0.014 0.022 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 170 401 170 401 170 401 n (Patient) 21 129 18 132 17 133 sCr only Median 64.7 111 67.7 109 68.2 106 Average 81.1 121 84.1 119 85.2 119 Stdev 44.6 67.9 44.9 68.0 45.4 68.0 p (t-test) 0.0039 0.012 0.019 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 174 401 174 401 174 401 n (Patient) 28 122 26 124 25 125 UO only Median 88.6 120 90.0 118 90.0 113 Average 96.7 131 97.1 129 97.5 128 Stdev 52.3 73.9 52.2 73.4 52.9 72.8 p (t-test) 0.0016 0.0039 0.0061 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 71 74 66 79 64 81 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.68 0.64 0.70 0.66 0.63 0.69 0.65 0.62 SE 0.052 0.051 0.046 0.059 0.054 0.046 0.061 0.056 0.046 p Value 8.7E−6 3.2E−4 0.0024 7.8E−4 0.0033 0.0061 0.0022 0.0071 0.0094 nCohort Recovered 21 28 71 18 26 66 17 25 64 nCohort Non-recovered 129 122 74 132 124 79 133 125 81 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 80% 80% 84% 78% 79% 81% 77% 78% 81% Specificity 57% 50% 34% 50% 46% 32% 47% 44% 33% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 53% 54% 59% 52% 53% 57% 52% 53% 56% Specificity 71% 68% 59% 67% 65% 58% 65% 64% 56% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 29% 29% 34% 28% 28% 33% 28% 28% 32% Specificity 95% 89% 83% 94% 88% 83% 94% 88% 83% OR Quartile 2 5.28 4.08 2.64 3.55 3.23 1.99 3.05 2.85 2.15 p Value 7.3E−4 0.0014 0.016 0.014 0.0093 0.077 0.035 0.022 0.050 Lower limit of 95% CI 2.01 1.72 1.20 1.29 1.33 0.927 1.08 1.16 0.999 Upper limit of 95% CI 13.9 9.70 5.81 9.77 7.82 4.28 8.60 7.00 4.62 OR Quartile 3 2.88 2.49 2.12 2.19 2.15 1.80 1.98 1.99 1.61 p Value 0.040 0.040 0.026 0.14 0.089 0.082 0.20 0.13 0.16 Lower limit of 95% CI 1.05 1.04 1.09 0.776 0.890 0.928 0.691 0.818 0.830 Upper limit of 95% CI 7.88 5.94 4.12 6.18 5.19 3.48 5.66 4.84 3.11 OR Quartile 4 8.04 3.35 2.51 6.62 3.01 2.45 6.17 2.85 2.28 p Value 0.046 0.060 0.022 0.071 0.087 0.028 0.083 0.11 0.044 Lower limit of 95% CI 1.04 0.951 1.14 0.850 0.851 1.10 0.789 0.803 1.02 Upper limit of 95% CI 62.1 11.8 5.50 51.5 10.7 5.46 48.2 10.1 5.07

TABLE 10.8 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 76.1 106 76.1 106 84.0 105 Average 90.2 120 90.3 120 91.9 119 Stdev 51.3 68.1 52.3 67.8 54.0 67.5 p (t-test) 0.022 0.029 0.057 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 246 401 246 401 246 401 n (Patient) 32 117 30 119 27 122 sCr only Median 86.1 109 87.0 106 87.0 106 Average 95.9 121 97.0 120 97.0 120 Stdev 51.8 69.1 52.1 69.1 52.1 69.1 p (t-test) 0.047 0.067 0.067 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 246 401 246 401 246 401 n (Patient) 38 110 37 111 37 111 UO only Median 98.3 118 96.1 115 106 108 Average 105 130 106 128 111 123 Stdev 56.9 73.2 58.0 72.1 58.5 71.9 p (t-test) 0.034 0.050 0.28 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 260 401 260 401 260 401 n (Patient) 64 69 61 72 55 78 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.60 0.60 0.64 0.60 0.59 0.62 0.60 0.54 SE 0.052 0.051 0.049 0.053 0.052 0.049 0.056 0.052 0.051 p Value 0.0073 0.041 0.046 0.0095 0.067 0.060 0.027 0.067 0.41 nCohort Recovered 32 38 64 30 37 61 27 37 55 nCohort Non-recovered 117 110 69 119 111 72 122 111 78 Cutoff Quartile 2 66.7 66.8 69.2 66.7 66.8 69.2 66.7 66.8 69.2 Sensitivity 79% 80% 81% 79% 79% 81% 79% 79% 76% Specificity 41% 39% 31% 40% 38% 31% 41% 38% 25% Cutoff Quartile 3 101 101 106 101 101 106 101 101 106 Sensitivity 53% 53% 54% 53% 52% 53% 52% 52% 50% Specificity 59% 58% 53% 60% 57% 52% 59% 57% 49% Cutoff Quartile 4 142 143 148 142 143 148 142 143 148 Sensitivity 29% 27% 33% 29% 27% 32% 28% 27% 29% Specificity 88% 82% 83% 87% 81% 82% 85% 81% 80% OR Quartile 2 2.65 2.61 1.96 2.51 2.33 1.87 2.54 2.33 1.06 p Value 0.022 0.019 0.10 0.035 0.040 0.12 0.038 0.040 0.89 Lower limit of 95% CI 1.15 1.17 0.878 1.07 1.04 0.845 1.05 1.04 0.478 Upper limit of 95% CI 6.12 5.81 4.37 5.88 5.22 4.16 6.13 5.22 2.35 OR Quartile 3 1.65 1.53 1.31 1.69 1.44 1.23 1.61 1.44 0.964 p Value 0.22 0.26 0.44 0.21 0.34 0.55 0.27 0.34 0.92 Lower limit of 95% CI 0.745 0.728 0.663 0.747 0.679 0.623 0.689 0.679 0.484 Upper limit of 95% CI 3.64 3.23 2.59 3.81 3.04 2.44 3.74 3.04 1.92 OR Quartile 4 2.87 1.66 2.41 2.60 1.59 2.13 2.22 1.59 1.67 p Value 0.066 0.28 0.036 0.096 0.33 0.070 0.17 0.33 0.22 Lower limit of 95% CI 0.935 0.661 1.06 0.844 0.631 0.940 0.715 0.631 0.736 Upper limit of 95% CI 8.80 4.17 5.47 8.01 4.00 4.84 6.90 4.00 3.80

TABLE 10.9 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 77.9 118 84.0 113 87.0 113 Average 88.8 125 91.7 123 94.1 121 Stdev 49.9 68.6 52.1 68.3 52.5 68.3 p (t-test) 0.0033 0.018 0.041 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 38 106 33 111 31 113 sCr only Median 85.2 118 85.2 118 87.0 113 Average 94.3 125 94.3 125 96.1 124 Stdev 50.5 70.1 50.5 70.1 50.7 70.2 p (t-test) 0.0088 0.0088 0.021 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 45 99 45 99 43 101 UO only Median 109 118 119 104 111 113 Average 111 131 119 124 116 125 Stdev 57.6 73.3 57.6 72.9 58.8 71.0 p (t-test) 0.091 0.67 0.46 Min 24.0 13.2 24.0 13.2 24.0 13.2 Max 260 401 260 401 260 401 n (Patient) 60 65 52 73 47 79 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.63 0.58 0.64 0.63 0.50 0.62 0.61 0.53 SE 0.048 0.048 0.051 0.052 0.048 0.053 0.054 0.049 0.053 p Value 4.2E−4 0.0062 0.13 0.0068 0.0062 0.99 0.025 0.020 0.61 nCohort Recovered 38 45 60 33 45 52 31 43 47 nCohort Non-recovered 106 99 65 111 99 73 113 101 79 Cutoff Quartile 2 67.0 67.0 75.7 67.0 67.0 75.7 67.0 67.0 75.8 Sensitivity 81% 81% 83% 78% 81% 75% 78% 80% 76% Specificity 42% 38% 33% 36% 38% 25% 35% 37% 28% Cutoff Quartile 3 103 103 113 103 103 113 103 103 112 Sensitivity 55% 55% 52% 53% 55% 48% 52% 53% 51% Specificity 63% 60% 52% 61% 60% 46% 58% 58% 51% Cutoff Quartile 4 146 146 152 146 146 152 146 146 152 Sensitivity 29% 28% 32% 28% 28% 29% 27% 28% 28% Specificity 87% 82% 82% 85% 82% 79% 84% 81% 79% OR Quartile 2 3.13 2.56 2.45 2.07 2.56 1.02 1.94 2.40 1.21 p Value 0.0056 0.019 0.037 0.090 0.019 0.97 0.13 0.030 0.65 Lower limit of 95% CI 1.40 1.17 1.06 0.893 1.17 0.447 0.820 1.09 0.531 Upper limit of 95% CI 7.01 5.59 5.70 4.80 5.59 2.32 4.57 5.28 2.75 OR Quartile 3 2.07 1.80 1.17 1.75 1.80 0.789 1.51 1.60 1.07 p Value 0.061 0.11 0.66 0.17 0.11 0.52 0.31 0.20 0.85 Lower limit of 95% CI 0.967 0.880 0.581 0.791 0.880 0.387 0.678 0.776 0.520 Upper limit of 95% CI 4.44 3.68 2.37 3.85 3.68 1.61 3.38 3.28 2.20 OR Quartile 4 2.73 1.82 2.13 2.17 1.82 1.51 1.97 1.68 1.43 p Value 0.056 0.18 0.077 0.14 0.18 0.34 0.20 0.25 0.41 Lower limit of 95% CI 0.974 0.756 0.922 0.769 0.756 0.652 0.693 0.694 0.608 Upper limit of 95% CI 7.64 4.40 4.90 6.13 4.40 3.47 5.58 4.06 3.36

TABLE 10.10 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 91.4 113 90.0 115 90.0 115 Average 101 128 100.0 128 100.0 128 Stdev 52.9 76.1 53.0 75.6 53.0 75.6 p (t-test) 0.010 0.0078 0.0078 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 81 69 80 70 80 70 sCr only Median 87.0 118 85.2 119 85.2 119 Average 94.1 129 92.9 129 92.9 129 Stdev 49.4 73.3 49.6 72.5 49.6 72.5 p (t-test) 0.0012 7.6E−4 7.6E−4 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 67 83 65 85 65 85 UO only Median 94.3 115 94.2 118 91.4 120 Average 102 134 101 134 100 135 Stdev 54.0 79.2 54.4 77.7 54.6 76.4 p (t-test) 0.0034 0.0026 0.0017 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 260 401 260 401 260 401 n (Patient) 95 54 93 56 91 58 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.60 0.64 0.62 0.61 0.65 0.63 0.61 0.65 0.64 SE 0.047 0.045 0.049 0.046 0.044 0.048 0.046 0.044 0.047 p Value 0.031 0.0020 0.016 0.020 7.6E−4 0.0091 0.020 7.6E−4 0.0039 nCohort Recovered 81 67 95 80 65 93 80 65 91 nCohort Non-recovered 69 83 54 70 85 56 70 85 58 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 81% 84% 83% 81% 85% 84% 81% 85% 84% Specificity 31% 37% 29% 31% 38% 30% 31% 38% 31% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 57% 57% 57% 57% 58% 59% 57% 58% 60% Specificity 56% 58% 54% 56% 60% 55% 56% 60% 56% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 33% 33% 37% 34% 33% 38% 34% 33% 38% Specificity 81% 84% 81% 82% 85% 82% 82% 85% 82% OR Quartile 2 1.92 3.21 2.09 1.99 3.46 2.25 1.99 3.46 2.42 p Value 0.094 0.0031 0.086 0.078 0.0017 0.058 0.078 0.0017 0.039 Lower limit of 95% CI 0.894 1.48 0.901 0.927 1.60 0.972 0.927 1.60 1.05 Upper limit of 95% CI 4.14 6.93 4.84 4.29 7.50 5.21 4.29 7.50 5.60 OR Quartile 3 1.62 1.82 1.56 1.71 2.04 1.74 1.71 2.04 1.94 p Value 0.14 0.072 0.19 0.10 0.033 0.10 0.10 0.033 0.052 Lower limit of 95% CI 0.851 0.948 0.797 0.897 1.06 0.891 0.897 1.06 0.993 Upper limit of 95% CI 3.10 3.49 3.06 3.28 3.94 3.41 3.28 3.94 3.79 OR Quartile 4 2.20 2.45 2.52 2.46 2.70 2.68 2.46 2.70 2.86 p Value 0.040 0.026 0.016 0.020 0.016 0.010 0.020 0.016 0.0064 Lower limit of 95% CI 1.04 1.11 1.18 1.15 1.20 1.26 1.15 1.20 1.34 Upper limit of 95% CI 4.66 5.42 5.35 5.25 6.08 5.70 5.25 6.08 6.11

Example 11. Use of Cathepsin B for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Cathepsin B is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 11.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.04 5.92 2.13 5.39 2.06 4.92 Average 2.93 14.6 3.03 14.4 2.98 14.3 Stdev 3.31 22.4 3.38 22.2 3.42 22.2 p (t-test) 0.0029 0.0047 0.0054 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 14.6 111 14.6 111 14.6 111 n (Patient) 34 114 32 116 31 117 sCr only Median 1.57 6.58 1.57 6.58 1.55 6.50 Average 2.86 16.0 2.86 16.0 2.82 15.9 Stdev 3.18 23.2 3.18 23.2 3.20 23.1 p (t-test) 2.0E−4 2.0E−4 2.4E−4 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 14.6 111 14.6 111 14.6 111 n (Patient) 46 102 46 102 45 103 UO only Median 3.05 5.36 3.29 4.83 3.17 4.85 Average 9.39 14.8 9.61 14.5 9.07 14.9 Stdev 16.2 23.9 16.3 23.6 15.8 23.8 p (t-test) 0.11 0.15 0.080 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 77 70 75 72 74 73 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.72 0.57 0.68 0.72 0.56 0.68 0.72 0.57 SE 0.048 0.042 0.047 0.050 0.042 0.047 0.050 0.042 0.047 p Value 9.4E−5 2.0E−7 0.13 4.2E−4 2.0E−7 0.23 2.6E−4 1.0E−7 0.14 nCohort Recovered 34 46 77 32 46 75 31 45 74 nCohort Non-recovered 114 102 70 116 102 72 117 103 73 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 76% 80% 76% 76% 80% 75% 76% 81% 75% Specificity 29% 37% 26% 28% 37% 25% 29% 38% 26% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 57% 60% 57% 56% 60% 56% 56% 60% 56% Specificity 74% 72% 56% 72% 72% 55% 74% 73% 55% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 31% 34% 27% 30% 34% 26% 30% 34% 27% Specificity 94% 96% 77% 94% 96% 76% 94% 96% 77% OR Quartile 2 1.34 2.40 1.09 1.23 2.40 1.02 1.30 2.52 1.06 p Value 0.50 0.026 0.81 0.65 0.026 0.96 0.56 0.020 0.89 Lower limit of 95% CI 0.571 1.11 0.518 0.510 1.11 0.483 0.537 1.16 0.501 Upper limit of 95% CI 3.16 5.21 2.31 2.97 5.21 2.14 3.15 5.47 2.22 OR Quartile 3 3.68 3.78 1.69 3.26 3.78 1.51 3.72 4.16 1.59 p Value 0.0026 5.5E−4 0.12 0.0067 5.5E−4 0.22 0.0036 2.8E−4 0.16 Lower limit of 95% CI 1.58 1.78 0.878 1.39 1.78 0.787 1.54 1.93 0.830 Upper limit of 95% CI 8.60 8.03 3.24 7.65 8.03 2.89 9.00 8.98 3.05 OR Quartile 4 7.09 11.5 1.22 6.48 11.5 1.14 6.19 11.1 1.27 p Value 0.0096 0.0012 0.60 0.014 0.0012 0.74 0.016 0.0014 0.54 Lower limit of 95% CI 1.61 2.63 0.579 1.47 2.63 0.539 1.40 2.53 0.599 Upper limit of 95% CI 31.2 50.2 2.57 28.6 50.2 2.39 27.4 48.4 2.67

TABLE 11.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.02 6.53 2.02 6.50 2.02 6.50 Average 3.19 16.0 3.21 15.7 3.21 15.7 Stdev 3.76 23.4 3.82 23.2 3.82 23.2 p (t-test) 2.8E−4 4.5E−4 4.5E−4 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 17.5 111 17.5 111 17.5 111 n (Patient) 47 101 45 103 45 103 sCr only Median 1.74 7.50 1.60 7.19 1.60 7.19 Average 3.05 17.0 3.01 16.9 3.01 16.9 Stdev 3.60 23.9 3.63 23.8 3.63 23.8 p (t-test) 3.6E−5 4.4E−5 4.4E−5 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 17.5 111 17.5 111 17.5 111 n (Patient) 54 94 53 95 53 95 UO only Median 3.04 5.87 3.04 5.87 3.04 5.87 Average 8.93 16.0 8.51 16.3 8.51 16.3 Stdev 15.7 24.9 15.2 24.9 15.2 24.9 p (t-test) 0.039 0.023 0.023 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 80 63 78 65 78 65 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.73 0.59 0.70 0.74 0.59 0.70 0.74 0.59 SE 0.044 0.041 0.048 0.044 0.041 0.048 0.044 0.041 0.048 p Value 4.0E−6 1.3E−8 0.072 8.0E−6 7.1E−9 0.061 8.0E−6 7.1E−9 0.061 nCohort Recovered 47 54 80 45 53 78 45 53 78 nCohort Non-recovered 101 94 63 103 95 65 103 95 65 Cutoff Quartile 2 1.13 1.13 1.06 1.13 1.13 1.06 1.13 1.13 1.06 Sensitivity 79% 82% 79% 79% 82% 78% 79% 82% 78% Specificity 34% 37% 29% 33% 38% 28% 33% 38% 28% Cutoff Quartile 3 3.63 3.63 3.80 3.63 3.63 3.80 3.63 3.63 3.80 Sensitivity 60% 63% 57% 60% 63% 57% 60% 63% 57% Specificity 72% 72% 55% 73% 74% 55% 73% 74% 55% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 34% 36% 30% 33% 36% 31% 33% 36% 31% Specificity 94% 94% 79% 93% 94% 79% 93% 94% 79% OR Quartile 2 1.97 2.66 1.55 1.84 2.78 1.43 1.84 2.78 1.43 p Value 0.086 0.012 0.27 0.12 0.0087 0.36 0.12 0.0087 0.36 Lower limit of 95% CI 0.909 1.24 0.712 0.845 1.30 0.663 0.845 1.30 0.663 Upper limit of 95% CI 4.25 5.71 3.38 4.01 5.97 3.09 4.01 5.97 3.09 OR Quartile 3 3.99 4.38 1.63 4.16 4.78 1.62 4.16 4.78 1.62 p Value 3.2E−4 6.9E−5 0.15 2.8E−4 3.4E−5 0.15 2.8E−4 3.4E−5 0.15 Lower limit of 95% CI 1.88 2.12 0.838 1.93 2.28 0.836 1.93 2.28 0.836 Upper limit of 95% CI 8.47 9.07 3.17 8.98 10.0 3.15 8.98 10.0 3.15 OR Quartile 4 7.44 9.63 1.60 6.90 9.29 1.72 6.90 9.29 1.72 p Value 0.0015 3.4E−4 0.22 0.0023 4.2E−4 0.16 0.0023 4.2E−4 0.16 Lower limit of 95% CI 2.15 2.79 0.749 1.99 2.69 0.804 1.99 2.69 0.804 Upper limit of 95% CI 25.7 33.2 3.42 23.9 32.0 3.69 23.9 32.0 3.69

TABLE 11.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.10 6.53 2.10 6.53 2.13 6.52 Average 5.53 16.4 5.53 16.4 5.61 16.2 Stdev 10.1 24.2 10.1 24.2 10.1 24.1 p (t-test) 0.0012 0.0012 0.0017 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 60 87 60 87 59 88 sCr only Median 2.10 7.37 2.10 7.37 2.10 7.37 Average 5.35 17.3 5.35 17.3 5.35 17.3 Stdev 9.78 24.6 9.78 24.6 9.78 24.6 p (t-test) 3.7E−4 3.7E−4 3.7E−4 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 64 82 64 82 64 82 UO only Median 3.61 6.53 3.61 6.53 3.61 5.87 Average 9.24 17.3 9.24 17.3 9.42 16.9 Stdev 15.8 26.0 15.8 26.0 16.0 25.7 p (t-test) 0.029 0.029 0.045 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 74 57 74 57 72 59 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.69 0.60 0.67 0.69 0.60 0.67 0.69 0.59 SE 0.044 0.043 0.050 0.044 0.043 0.050 0.044 0.043 0.050 p Value 8.8E−5 6.9E−6 0.058 8.8E−5 6.9E−6 0.058 1.9E−4 6.9E−6 0.082 nCohort Recovered 60 64 74 60 64 74 59 64 72 nCohort Non-recovered 87 82 57 87 82 57 88 82 59 Cutoff Quartile 2 1.13 1.12 1.16 1.13 1.12 1.16 1.13 1.12 1.16 Sensitivity 80% 83% 79% 80% 83% 79% 80% 83% 78% Specificity 33% 36% 28% 33% 36% 28% 32% 36% 28% Cutoff Quartile 3 3.62 3.63 4.38 3.62 3.63 4.38 3.62 3.63 4.38 Sensitivity 63% 65% 56% 63% 65% 56% 62% 65% 56% Specificity 68% 69% 54% 68% 69% 54% 68% 69% 54% Cutoff Quartile 4 13.1 13.3 14.2 13.1 13.3 14.2 13.1 13.3 14.2 Sensitivity 33% 35% 30% 33% 35% 30% 33% 35% 29% Specificity 87% 88% 78% 87% 88% 78% 86% 88% 78% OR Quartile 2 2.06 2.72 1.49 2.06 2.72 1.49 1.85 2.72 1.36 p Value 0.061 0.011 0.34 0.061 0.011 0.34 0.11 0.011 0.45 Lower limit of 95% CI 0.968 1.26 0.659 0.968 1.26 0.659 0.870 1.26 0.610 Upper limit of 95% CI 4.38 5.88 3.35 4.38 5.88 3.35 3.92 5.88 3.04 OR Quartile 3 3.71 4.02 1.51 3.71 4.02 1.51 3.51 4.02 1.50 p Value 2.3E−4 8.9E−5 0.25 2.3E−4 8.9E−5 0.25 4.1E−4 8.9E−5 0.25 Lower limit of 95% CI 1.85 2.00 0.752 1.85 2.00 0.752 1.75 2.00 0.751 Upper limit of 95% CI 7.45 8.06 3.02 7.45 8.06 3.02 7.04 8.06 3.00 OR Quartile 4 3.25 3.83 1.54 3.25 3.83 1.54 3.13 3.83 1.42 p Value 0.0078 0.0024 0.29 0.0078 0.0024 0.29 0.0099 0.0024 0.39 Lower limit of 95% CI 1.36 1.61 0.697 1.36 1.61 0.697 1.32 1.61 0.642 Upper limit of 95% CI 7.74 9.13 3.40 7.74 9.13 3.40 7.46 9.13 3.13

TABLE 11.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.17 7.97 2.20 6.94 2.17 7.00 Average 5.61 18.2 5.77 17.5 5.70 17.2 Stdev 9.71 25.6 9.89 25.3 10.0 24.9 p (t-test) 2.1E−4 5.6E−4 8.4E−4 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 68 74 65 77 62 80 sCr only Median 2.17 8.29 2.17 8.29 2.13 8.44 Average 5.56 18.6 5.56 18.6 5.43 18.5 Stdev 9.58 25.8 9.58 25.8 9.59 25.7 p (t-test) 1.2E−4 1.2E−4 1.1E−4 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 70 72 70 72 69 73 UO only Median 3.61 7.81 3.62 6.20 3.63 6.20 Average 9.26 18.2 9.10 17.6 9.35 17.1 Stdev 15.9 26.8 15.6 26.2 15.7 25.9 p (t-test) 0.024 0.030 0.046 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 70 51 65 56 64 58 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.69 0.61 0.67 0.69 0.60 0.67 0.70 0.58 SE 0.044 0.044 0.052 0.045 0.044 0.052 0.045 0.044 0.052 p Value 2.5E−5 1.3E−5 0.032 1.3E−4 1.3E−5 0.062 1.3E−4 4.9E−6 0.12 nCohort Recovered 68 70 70 65 70 65 62 69 64 nCohort Non-recovered 74 72 51 77 72 56 80 73 58 Cutoff Quartile 2 1.12 1.12 1.22 1.12 1.12 1.22 1.12 1.12 1.23 Sensitivity 82% 83% 80% 81% 83% 79% 80% 84% 76% Specificity 34% 34% 29% 32% 34% 28% 32% 35% 27% Cutoff Quartile 3 3.71 3.71 4.38 3.71 3.71 4.38 3.71 3.71 4.56 Sensitivity 65% 65% 59% 64% 65% 57% 64% 66% 57% Specificity 66% 66% 56% 66% 66% 55% 68% 67% 56% Cutoff Quartile 4 13.3 13.3 16.1 13.3 13.3 16.1 13.3 13.3 15.7 Sensitivity 36% 38% 31% 35% 38% 30% 35% 38% 29% Specificity 87% 87% 79% 86% 87% 78% 87% 88% 78% OR Quartile 2 2.40 2.61 1.64 1.97 2.61 1.40 1.90 2.71 1.14 p Value 0.028 0.018 0.26 0.083 0.018 0.43 0.098 0.014 0.76 Lower limit of 95% CI 1.10 1.18 0.691 0.916 1.18 0.607 0.887 1.23 0.502 Upper limit of 95% CI 5.24 5.76 3.89 4.25 5.76 3.25 4.09 5.99 2.58 OR Quartile 3 3.61 3.60 1.80 3.42 3.60 1.66 3.69 3.84 1.70 p Value 2.8E−4 2.8E−4 0.12 5.0E−4 2.8E−4 0.17 2.6E−4 1.5E−4 0.15 Lower limit of 95% CI 1.81 1.80 0.866 1.71 1.80 0.805 1.83 1.91 0.829 Upper limit of 95% CI 7.22 7.20 3.73 6.84 7.20 3.40 7.44 7.70 3.48 OR Quartile 4 3.77 4.07 1.68 3.36 4.07 1.59 3.63 4.74 1.48 p Value 0.0021 0.0012 0.22 0.0050 0.0012 0.27 0.0038 4.9E−4 0.35 Lower limit of 95% CI 1.62 1.74 0.737 1.44 1.74 0.699 1.52 1.98 0.653 Upper limit of 95% CI 8.78 9.49 3.81 7.82 9.49 3.61 8.70 11.4 3.36

TABLE 11.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.46 7.97 2.42 7.81 2.46 7.17 Average 8.00 18.4 8.04 18.1 8.12 17.8 Stdev 14.8 25.8 14.9 25.7 15.0 25.5 p (t-test) 0.0023 0.0030 0.0042 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 92 56 91 57 90 58 sCr only Median 2.22 8.53 2.22 8.53 2.22 8.53 Average 5.57 19.8 5.57 19.8 5.57 19.8 Stdev 9.15 26.7 9.15 26.7 9.15 26.7 p (t-test) 1.3E−5 1.3E−5 1.3E−5 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 57.5 111 57.5 111 57.5 111 n (Patient) 82 66 82 66 82 66 UO only Median 3.03 7.97 3.03 7.97 3.03 7.17 Average 8.64 18.9 8.64 18.9 8.76 18.2 Stdev 14.9 27.4 14.9 27.4 15.0 27.0 p (t-test) 0.0039 0.0039 0.0070 Min 0.0435 0.126 0.0435 0.126 0.0435 0.126 Max 103 111 103 111 103 111 n (Patient) 99 48 99 48 97 50 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.70 0.62 0.65 0.70 0.62 0.64 0.70 0.61 SE 0.048 0.044 0.050 0.047 0.044 0.050 0.047 0.044 0.050 p Value 0.0018 8.3E−6 0.016 0.0016 8.3E−6 0.016 0.0035 8.3E−6 0.028 nCohort Recovered 92 82 99 91 82 99 90 82 97 nCohort Non-recovered 56 66 48 57 66 48 58 66 50 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 82% 83% 79% 82% 83% 79% 81% 83% 78% Specificity 29% 32% 27% 30% 32% 27% 29% 32% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 64% 67% 62% 65% 67% 62% 64% 67% 62% Specificity 59% 63% 56% 59% 63% 56% 59% 63% 56% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 36% 39% 33% 35% 39% 33% 34% 39% 32% Specificity 82% 87% 79% 81% 87% 79% 81% 87% 78% OR Quartile 2 1.91 2.32 1.42 1.98 2.32 1.42 1.74 2.32 1.30 p Value 0.12 0.038 0.40 0.10 0.038 0.40 0.18 0.038 0.53 Lower limit of 95% CI 0.843 1.05 0.624 0.876 1.05 0.624 0.781 1.05 0.580 Upper limit of 95% CI 4.33 5.15 3.25 4.49 5.15 3.25 3.86 5.15 2.91 OR Quartile 3 2.56 3.47 2.08 2.70 3.47 2.08 2.52 3.47 2.05 p Value 0.0073 3.5E−4 0.042 0.0046 3.5E−4 0.042 0.0077 3.5E−4 0.044 Lower limit of 95% CI 1.29 1.75 1.03 1.36 1.75 1.03 1.28 1.75 1.02 Upper limit of 95% CI 5.08 6.85 4.22 5.36 6.85 4.22 4.98 6.85 4.12 OR Quartile 4 2.45 4.20 1.86 2.35 4.20 1.86 2.26 4.20 1.70 p Value 0.021 4.8E−4 0.11 0.027 4.8E−4 0.11 0.035 4.8E−4 0.17 Lower limit of 95% CI 1.15 1.88 0.860 1.10 1.88 0.860 1.06 1.88 0.792 Upper limit of 95% CI 5.24 9.38 4.01 5.02 9.38 4.01 4.81 9.38 3.66

TABLE 11.6 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 67.7 109 67.7 109 67.1 111 Average 83.1 121 81.1 121 79.3 121 Stdev 43.4 68.6 41.1 68.5 40.7 68.2 p (t-test) 0.0032 0.0025 0.0018 Min 31.3 13.2 31.3 13.2 31.3 13.2 Max 174 401 170 401 170 401 n (Patient) 32 118 30 120 29 121 sCr only Median 67.1 113 67.1 113 66.9 115 Average 83.5 124 83.5 124 82.4 125 Stdev 44.4 69.2 44.4 69.2 44.4 68.9 p (t-test) 5.8E−4 5.8E−4 4.3E−4 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 182 401 182 401 182 401 n (Patient) 41 109 41 109 40 110 UO only Median 94.2 113 94.2 113 94.2 112 Average 102 126 101 126 101 125 Stdev 53.7 75.0 53.4 74.6 53.8 74.2 p (t-test) 0.023 0.023 0.025 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 76 73 74 75 73 76 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.69 0.59 0.69 0.69 0.59 0.70 0.69 0.59 SE 0.049 0.046 0.047 0.050 0.046 0.046 0.049 0.045 0.046 p Value 3.4E−4 4.2E−5 0.050 1.6E−4 4.2E−5 0.047 5.5E−5 1.7E−5 0.048 nCohort Recovered 32 41 76 30 41 74 29 40 73 nCohort Non-recovered 118 109 73 120 109 75 121 110 76 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 81% 83% 81% 80% 83% 80% 80% 83% 80% Specificity 47% 46% 30% 47% 46% 30% 48% 48% 30% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 53% 56% 56% 53% 56% 56% 54% 56% 55% Specificity 62% 66% 55% 63% 66% 55% 66% 68% 55% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 30% 30% 32% 30% 30% 32% 30% 30% 32% Specificity 91% 88% 80% 93% 88% 81% 93% 88% 81% OR Quartile 2 3.64 4.09 1.83 3.50 4.09 1.69 3.77 4.33 1.75 p Value 0.0023 4.6E−4 0.12 0.0037 4.6E−4 0.17 0.0023 2.9E−4 0.14 Lower limit of 95% CI 1.59 1.86 0.854 1.50 1.86 0.796 1.61 1.96 0.825 Upper limit of 95% CI 8.36 9.00 3.91 8.15 9.00 3.60 8.87 9.58 3.73 OR Quartile 3 1.91 2.45 1.58 1.97 2.45 1.58 2.21 2.68 1.50 p Value 0.11 0.019 0.16 0.11 0.019 0.16 0.067 0.011 0.22 Lower limit of 95% CI 0.856 1.16 0.829 0.865 1.16 0.828 0.947 1.25 0.785 Upper limit of 95% CI 4.26 5.18 3.02 4.50 5.18 3.02 5.13 5.74 2.86 OR Quartile 4 4.08 3.13 1.87 6.00 3.13 2.02 5.72 3.00 1.95 p Value 0.028 0.029 0.10 0.018 0.029 0.070 0.022 0.035 0.085 Lower limit of 95% CI 1.16 1.13 0.883 1.36 1.13 0.946 1.29 1.08 0.912 Upper limit of 95% CI 14.3 8.68 3.96 26.5 8.68 4.30 25.3 8.34 4.15

TABLE 11.7 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 68.2 116 67.1 118 67.1 118 Average 83.7 126 80.5 126 80.5 126 Stdev 42.8 70.0 40.8 69.5 40.8 69.5 p (t-test) 2.4E−4 7.8E−5 7.8E−5 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 174 401 171 401 171 401 n (Patient) 45 105 43 107 43 107 sCr only Median 71.0 117 68.2 118 68.2 118 Average 86.0 127 85.0 127 85.0 127 Stdev 44.4 70.6 44.4 70.3 44.4 70.3 p (t-test) 2.6E−4 2.0E−4 2.0E−4 Min 23.6 13.2 23.6 13.2 23.6 13.2 Max 182 401 182 401 182 401 n (Patient) 50 100 49 101 49 101 UO only Median 88.6 120 87.0 120 87.0 120 Average 98.7 133 97.8 133 97.8 133 Stdev 54.1 74.8 54.1 73.9 54.1 73.9 p (t-test) 0.0018 0.0012 0.0012 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 79 66 77 68 77 68 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.68 0.64 0.71 0.69 0.65 0.71 0.69 0.65 SE 0.045 0.044 0.046 0.044 0.044 0.046 0.044 0.044 0.046 p Value 2.3E−5 5.2E−5 0.0028 1.4E−6 2.5E−5 0.0014 1.4E−6 2.5E−5 0.0014 nCohort Recovered 45 50 79 43 49 77 43 49 77 nCohort Non-recovered 105 100 66 107 101 68 107 101 68 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 83% 84% 85% 83% 84% 85% 83% 84% 85% Specificity 44% 44% 33% 47% 45% 34% 47% 45% 34% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 57% 58% 61% 58% 58% 60% 58% 58% 60% Specificity 67% 66% 58% 70% 67% 58% 70% 67% 58% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 31% 31% 35% 32% 31% 35% 32% 31% 35% Specificity 89% 86% 82% 91% 86% 83% 91% 86% 83% OR Quartile 2 3.87 4.12 2.75 4.30 4.33 2.96 4.30 4.33 2.96 p Value 6.4E−4 3.3E−4 0.016 2.7E−4 2.1E−4 0.0096 2.7E−4 2.1E−4 0.0096 Lower limit of 95% CI 1.78 1.90 1.21 1.96 1.99 1.30 1.96 1.99 1.30 Upper limit of 95% CI 8.41 8.94 6.24 9.42 9.41 6.72 9.42 9.41 6.72 OR Quartile 3 2.67 2.68 2.14 3.18 2.90 2.14 3.18 2.90 2.14 p Value 0.0085 0.0063 0.025 0.0027 0.0036 0.025 0.0027 0.0036 0.025 Lower limit of 95% CI 1.28 1.32 1.10 1.49 1.42 1.10 1.49 1.42 1.10 Upper limit of 95% CI 5.54 5.44 4.17 6.77 5.93 4.15 6.77 5.93 4.15 OR Quartile 4 3.67 2.76 2.48 4.54 2.66 2.69 4.54 2.66 2.69 p Value 0.012 0.028 0.020 0.0074 0.034 0.013 0.0074 0.034 0.013 Lower limit of 95% CI 1.33 1.12 1.15 1.50 1.07 1.24 1.50 1.07 1.24 Upper limit of 95% CI 10.1 6.82 5.35 13.7 6.57 5.84 13.7 6.57 5.84

TABLE 11.8 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 80.0 120 80.0 120 80.3 119 Average 90.7 128 90.7 128 91.3 128 Stdev 49.3 71.0 49.3 71.0 49.5 71.0 p (t-test) 5.5E−4 5.5E−4 9.3E−4 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 58 91 58 91 57 92 sCr only Median 85.2 119 85.2 119 85.2 119 Average 95.4 127 95.4 127 95.4 127 Stdev 50.6 72.1 50.6 72.1 50.6 72.1 p (t-test) 0.0034 0.0034 0.0034 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 61 87 61 87 61 87 UO only Median 94.2 129 94.2 129 94.2 122 Average 103 136 103 136 104 134 Stdev 56.0 74.2 56.0 74.2 56.5 73.8 p (t-test) 0.0049 0.0049 0.0082 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 260 401 260 401 260 401 n (Patient) 72 61 72 61 70 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.63 0.64 0.67 0.63 0.64 0.66 0.63 0.63 SE 0.044 0.046 0.049 0.044 0.046 0.049 0.045 0.046 0.049 p Value 1.6E−4 0.0037 0.0051 1.6E−4 0.0037 0.0051 3.5E−4 0.0037 0.0086 nCohort Recovered 58 61 72 58 61 72 57 61 70 nCohort Non-recovered 91 87 61 91 87 61 92 87 63 Cutoff Quartile 2 66.7 66.8 69.2 66.7 66.8 69.2 66.7 66.8 69.2 Sensitivity 84% 83% 87% 84% 83% 87% 83% 83% 86% Specificity 38% 36% 35% 38% 36% 35% 37% 36% 34% Cutoff Quartile 3 101 101 106 101 101 106 101 101 106 Sensitivity 57% 56% 57% 57% 56% 57% 57% 56% 57% Specificity 60% 59% 56% 60% 59% 56% 60% 59% 56% Cutoff Quartile 4 142 143 148 142 143 148 142 143 148 Sensitivity 33% 30% 36% 33% 30% 36% 33% 30% 35% Specificity 86% 82% 83% 86% 82% 83% 86% 82% 83% OR Quartile 2 3.10 2.71 3.52 3.10 2.71 3.52 2.77 2.71 3.13 p Value 0.0039 0.011 0.0054 0.0039 0.011 0.0054 0.0087 0.011 0.0094 Lower limit of 95% CI 1.44 1.26 1.45 1.44 1.26 1.45 1.29 1.26 1.32 Upper limit of 95% CI 6.67 5.81 8.56 6.67 5.81 8.56 5.94 5.81 7.41 OR Quartile 3 2.03 1.86 1.68 2.03 1.86 1.68 1.92 1.86 1.68 p Value 0.039 0.067 0.14 0.039 0.067 0.14 0.056 0.067 0.14 Lower limit of 95% CI 1.04 0.957 0.846 1.04 0.957 0.846 0.983 0.957 0.844 Upper limit of 95% CI 3.97 3.60 3.35 3.97 3.60 3.35 3.76 3.60 3.33 OR Quartile 4 3.07 1.94 2.82 3.07 1.94 2.82 2.96 1.94 2.59 p Value 0.011 0.10 0.012 0.011 0.10 0.012 0.014 0.10 0.021 Lower limit of 95% CI 1.29 0.872 1.25 1.29 0.872 1.25 1.25 0.872 1.15 Upper limit of 95% CI 7.30 4.30 6.34 7.30 4.30 6.34 7.04 4.30 5.82

TABLE 11.9 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 88.6 122 87.8 120 88.6 119 Average 96.6 131 96.1 130 96.1 129 Stdev 49.0 74.1 49.3 73.2 50.4 72.3 p (t-test) 0.0016 0.0019 0.0030 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 65 79 62 82 59 85 sCr only Median 91.4 122 91.4 122 92.9 120 Average 97.8 131 97.8 131 98.0 130 Stdev 49.1 74.8 49.1 74.8 49.5 74.5 p (t-test) 0.0024 0.0024 0.0032 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 67 77 67 77 66 78 UO only Median 103 122 106 118 103 118 Average 110 136 111 133 111 132 Stdev 55.0 77.5 56.3 75.3 56.8 74.4 p (t-test) 0.034 0.069 0.077 Min 24.0 13.2 24.0 13.2 24.0 13.2 Max 260 401 260 401 260 401 n (Patient) 70 55 65 60 64 62 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.63 0.59 0.64 0.63 0.58 0.63 0.62 0.58 SE 0.046 0.046 0.052 0.046 0.046 0.051 0.046 0.046 0.051 p Value 0.0031 0.0063 0.078 0.0027 0.0063 0.13 0.0036 0.0086 0.12 nCohort Recovered 65 67 70 62 67 65 59 66 64 nCohort Non-recovered 79 77 55 82 77 60 85 78 62 Cutoff Quartile 2 67.0 67.0 75.7 67.0 67.0 75.7 67.0 67.0 75.8 Sensitivity 82% 82% 84% 82% 82% 83% 82% 82% 82% Specificity 34% 33% 31% 34% 33% 32% 36% 33% 33% Cutoff Quartile 3 103 103 113 103 103 113 103 103 112 Sensitivity 56% 56% 55% 56% 56% 53% 55% 55% 53% Specificity 57% 57% 53% 58% 57% 52% 58% 56% 53% Cutoff Quartile 4 146 146 152 146 146 152 146 146 152 Sensitivity 33% 32% 36% 33% 32% 33% 32% 32% 32% Specificity 85% 84% 83% 85% 84% 82% 85% 83% 81% OR Quartile 2 2.38 2.20 2.34 2.29 2.20 2.39 2.58 2.29 2.26 p Value 0.028 0.045 0.056 0.035 0.045 0.046 0.016 0.036 0.055 Lower limit of 95% CI 1.10 1.02 0.977 1.06 1.02 1.01 1.19 1.06 0.983 Upper limit of 95% CI 5.15 4.76 5.62 4.93 4.76 5.61 5.58 4.95 5.22 OR Quartile 3 1.66 1.66 1.35 1.77 1.66 1.25 1.68 1.57 1.29 p Value 0.13 0.13 0.41 0.094 0.13 0.53 0.13 0.18 0.48 Lower limit of 95% CI 0.857 0.856 0.662 0.908 0.856 0.621 0.860 0.810 0.640 Upper limit of 95% CI 3.22 3.21 2.73 3.45 3.21 2.53 3.29 3.03 2.60 OR Quartile 4 2.70 2.45 2.76 2.89 2.45 2.21 2.59 2.36 2.06 p Value 0.018 0.029 0.016 0.014 0.029 0.060 0.027 0.036 0.085 Lower limit of 95% CI 1.19 1.10 1.20 1.24 1.10 0.968 1.11 1.06 0.906 Upper limit of 95% CI 6.13 5.47 6.33 6.72 5.47 5.04 6.01 5.27 4.70

TABLE 11.10 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 94.2 113 92.8 115 91.4 118 Average 101 131 101 130 101 131 Stdev 52.5 78.8 52.8 78.2 52.8 77.6 p (t-test) 0.0070 0.0068 0.0053 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 89 61 88 62 87 63 sCr only Median 91.4 118 91.4 118 91.4 118 Average 96.8 132 96.8 132 96.8 132 Stdev 48.9 76.9 48.9 76.9 48.9 76.9 p (t-test) 0.0010 0.0010 0.0010 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 246 401 n (Patient) 79 71 79 71 79 71 UO only Median 95.2 113 95.2 113 94.2 118 Average 103 134 103 134 102 134 Stdev 54.5 80.1 54.5 80.1 54.9 78.6 p (t-test) 0.0051 0.0051 0.0040 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 260 401 260 401 260 401 n (Patient) 98 51 98 51 96 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.60 0.63 0.61 0.60 0.63 0.61 0.61 0.63 0.62 SE 0.048 0.046 0.050 0.047 0.046 0.050 0.047 0.046 0.049 p Value 0.033 0.0045 0.028 0.029 0.0045 0.028 0.019 0.0045 0.016 nCohort Recovered 89 79 98 88 79 98 87 79 96 nCohort Non-recovered 61 71 51 62 71 51 63 71 53 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 82% 85% 84% 82% 85% 84% 83% 85% 85% Specificity 30% 34% 30% 31% 34% 30% 31% 34% 30% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 56% 56% 57% 56% 56% 57% 57% 56% 58% Specificity 54% 56% 53% 55% 56% 53% 55% 56% 54% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 34% 35% 35% 34% 35% 35% 35% 35% 36% Specificity 81% 84% 80% 81% 84% 80% 82% 84% 80% OR Quartile 2 1.98 2.83 2.26 2.05 2.83 2.26 2.13 2.83 2.43 p Value 0.092 0.010 0.067 0.076 0.010 0.067 0.062 0.010 0.045 Lower limit of 95% CI 0.895 1.28 0.946 0.928 1.28 0.946 0.962 1.28 1.02 Upper limit of 95% CI 4.38 6.26 5.39 4.54 6.26 5.39 4.70 6.26 5.81 OR Quartile 3 1.47 1.62 1.49 1.56 1.62 1.49 1.64 1.62 1.67 p Value 0.25 0.14 0.25 0.19 0.14 0.25 0.14 0.14 0.14 Lower limit of 95% CI 0.766 0.850 0.754 0.809 0.850 0.754 0.853 0.850 0.845 Upper limit of 95% CI 2.84 3.09 2.95 2.99 3.09 2.95 3.16 3.09 3.28 OR Quartile 4 2.22 2.76 2.13 2.14 2.76 2.13 2.38 2.76 2.26 p Value 0.036 0.0097 0.050 0.046 0.0097 0.050 0.023 0.0097 0.033 Lower limit of 95% CI 1.05 1.28 0.999 1.01 1.28 0.999 1.12 1.28 1.07 Upper limit of 95% CI 4.69 5.95 4.53 4.51 5.95 4.53 5.04 5.95 4.81

Example 12. Use of Tenascin for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Tenascin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 12.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 24200 53600 23000 53700 23000 53700 Average 30200 56900 26300 56900 26300 56900 Stdev 14500 26100 9010 26000 9010 26000 p (t-test) 0.0028 0.0011 0.0011 Min 20500 15600 20500 15600 20500 15600 Max 61600 141000 47900 141000 47900 141000 n (Patient) 9 141 8 142 8 142 sCr only Median 36100 53700 35600 53800 35600 53800 Average 44200 56800 43200 56800 43200 56800 Stdev 27100 25900 27600 25800 27600 25800 p (t-test) 0.057 0.044 0.044 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 18 132 17 133 17 133 UO only Median 46800 57200 45600 58200 46800 55700 Average 46100 61700 45900 61400 46100 60700 Stdev 20600 28200 20300 28100 20700 27900 p (t-test) 2.8E−4 3.3E−4 8.3E−4 Min 17100 15600 17100 15600 17100 15600 Max 97900 141000 97900 141000 97900 141000 n (Patient) 63 86 60 89 57 92 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.85 0.67 0.67 0.91 0.69 0.67 0.91 0.69 0.66 SE 0.048 0.061 0.044 0.034 0.060 0.044 0.034 0.060 0.044 p Value 3.8E−13 0.0047 1.2E−4 0 0.0014 9.6E−5 0 0.0014 2.8E−4 nCohort Recovered 9 18 63 8 17 60 8 17 57 nCohort 141 132 86 142 133 89 142 133 92 Non-recovered Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 78% 77% 83% 78% 77% 82% 78% 77% 82% Specificity 78% 44% 35% 88% 47% 35% 88% 47% 35% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 52% 53% 59% 53% 53% 60% 53% 53% 59% Specificity 89% 72% 62% 100%  76% 63% 100%  76% 63% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 27% 27% 33% 27% 27% 33% 27% 27% 32% Specificity 100%  89% 84% 100%  88% 85% 100%  88% 84% OR Quartile 2 12.4 2.72 2.54 25.1 3.05 2.46 25.1 3.05 2.38 p Value 0.0023 0.053 0.016 0.0031 0.035 0.020 0.0031 0.035 0.024 Lower limit of 2.45 0.986 1.19 2.97 1.08 1.15 2.97 1.08 1.12 95% CI Upper limit of 62.8 7.50 5.43 212 8.60 5.24 212 8.60 5.08 95% CI OR Quartile 3 8.84 2.94 2.37 19.0 3.72 2.54 19.0 3.72 2.44 p Value 0.042 0.052 0.011 0.044 0.028 0.0067 0.044 0.028 0.010 Lower limit of 1.08 0.990 1.22 1.08 1.15 1.30 1.08 1.15 1.23 95% CI Upper limit of 72.5 8.70 4.61 336 12.0 4.99 336 12.0 4.81 95% CI OR Quartile 4 7.07 3.00 2.56 6.26 2.78 2.74 6.26 2.78 2.46 p Value 0.18 0.16 0.023 0.21 0.19 0.018 0.21 0.19 0.035 Lower limit of 0.402 0.657 1.14 0.353 0.606 1.19 0.353 0.606 1.06 95% CI Upper limit of 124 13.7 5.77 111 12.8 6.32 111 12.8 5.67 95% CI

TABLE 12.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 29900 55100 26200 55100 25200 55100 Average 33900 58800 31900 58500 30300 58500 Stdev 13200 26200 12400 26100 10700 26000 p (t-test) 3.7E−5 3.9E−5 2.0E−5 Min 20500 15600 20500 15600 20500 15600 Max 61600 141000 59100 141000 51700 141000 n (Patient) 21 129 18 132 17 133 sCr only Median 36100 55100 35300 55100 35000 55100 Average 40900 58600 40300 58400 39500 58400 Stdev 23100 25900 23600 25800 23700 25700 p (t-test) 0.0012 0.0012 8.6E−4 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 28 122 26 124 25 125 UO only Median 44700 62700 44500 62000 44500 61600 Average 46300 64800 46100 63900 46100 63400 Stdev 19100 28900 19000 28700 19100 28500 p (t-test) 1.2E−5 3.1E−5 5.1E−5 Min 18800 15600 20500 15600 20500 15600 Max 97900 141000 97900 141000 97900 141000 n (Patient) 71 74 66 79 64 81 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.81 0.73 0.70 0.84 0.74 0.69 0.86 0.75 0.69 SE 0.041 0.047 0.043 0.039 0.047 0.043 0.036 0.046 0.043 p Value 5.1E−14 1.3E−6 4.2E−6 0 3.1E−7 7.0E−6 0 4.4E−8 1.0E−5 nCohort Recovered 21 28 71 18 26 66 17 25 64 nCohort 129 122 74 132 124 79 133 125 81 Non-recovered Cutoff Quartile 2 34800 34800 35000 34800 34800 35000 34800 34800 35000 Sensitivity 79% 79% 84% 80% 79% 84% 80% 79% 83% Specificity 52% 43% 34% 61% 46% 35% 65% 48% 34% Cutoff Quartile 3 52000 52000 52200 52000 52000 52200 52000 52000 52200 Sensitivity 57% 57% 65% 56% 56% 65% 56% 57% 64% Specificity 90% 79% 65% 94% 81% 67% 100%  84% 67% Cutoff Quartile 4 70700 70700 70800 70700 70700 70800 70700 70700 70800 Sensitivity 29% 30% 38% 29% 29% 37% 29% 29% 36% Specificity 100%  93% 87% 100%  92% 88% 100%  92% 88% OR Quartile 2 4.16 2.77 2.64 6.11 3.23 2.72 7.20 3.51 2.51 p Value 0.0035 0.021 0.016 6.3E−4 0.0093 0.012 3.4E−4 0.0059 0.020 Lower limit of 1.60 1.17 1.20 2.16 1.33 1.24 2.44 1.44 1.16 95% CI Upper limit of 10.8 6.58 5.81 17.3 7.82 5.93 21.2 8.61 5.43 95% CI OR Quartile 3 12.4 4.77 3.40 21.7 5.44 3.64 45.2 6.90 3.67 p Value 9.9E−4 0.0016 4.4E−4 0.0032 0.0014 2.3E−4 0.0084 7.7E−4 2.3E−4 Lower limit of 2.77 1.81 1.72 2.80 1.93 1.83 2.66 2.24 1.84 95% CI Upper limit of 55.4 12.6 6.72 168 15.4 7.25 767 21.3 7.33 95% CI OR Quartile 4 18.1 5.44 4.19 15.1 4.91 4.20 14.1 4.65 3.90 p Value 0.045 0.026 8.5E−4 0.061 0.037 0.0012 0.067 0.044 0.0021 Lower limit of 1.07 1.23 1.81 0.886 1.10 1.76 0.828 1.04 1.64 95% CI Upper limit of 306 24.1 9.74 256 21.9 10.0 241 20.8 9.31 95% CI

TABLE 12.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 35000 55200 34900 55800 34700 56000 Average 41700 59200 38500 59800 36800 59600 Stdev 23300 25800 18300 26300 17100 26200 p (t-test) 7.0E−4 4.8E−5 2.9E−5 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 90400 141000 90400 141000 n (Patient) 32 117 30 119 27 122 sCr only Median 36900 55500 36600 55800 36600 55800 Average 43500 59800 43100 59800 43100 59800 Stdev 23000 26100 23200 26000 23200 26000 p (t-test) 8.7E−4 7.1E−4 7.1E−4 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 38 110 37 111 37 111 UO only Median 47800 61600 47700 61200 47700 59500 Average 51200 63500 50200 63900 50500 62600 Stdev 20900 29200 19100 29700 19200 29300 p (t-test) 0.0063 0.0024 0.0079 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 97900 141000 97900 141000 n (Patient) 64 69 61 72 55 78 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.70 0.63 0.76 0.71 0.63 0.79 0.71 0.62 SE 0.045 0.045 0.048 0.043 0.045 0.048 0.042 0.045 0.049 p Value 4.4E−7 7.4E−6 0.0095 6.9E−10 3.1E−6 0.0053 6.3E−12 3.1E−6 0.015 nCohort Recovered 32 38 64 30 37 61 27 37 55 nCohort 117 110 69 119 111 72 122 111 78 Non-recovered Cutoff Quartile 2 35000 35000 37100 35000 35000 37100 35000 35000 37100 Sensitivity 81% 81% 78% 82% 81% 78% 81% 81% 77% Specificity 47% 42% 28% 50% 43% 28% 52% 43% 27% Cutoff Quartile 3 52200 52300 54300 52200 52300 54300 52200 52300 54300 Sensitivity 58% 58% 58% 59% 59% 58% 59% 59% 58% Specificity 78% 74% 58% 83% 76% 59% 89% 76% 60% Cutoff Quartile 4 70800 70900 71600 70800 70900 71600 70800 70900 71600 Sensitivity 29% 29% 35% 29% 29% 35% 30% 29% 33% Specificity 88% 87% 84% 90% 86% 85% 93% 86% 85% OR Quartile 2 3.81 3.08 1.41 4.41 3.27 1.35 4.64 3.27 1.25 p Value 0.0017 0.0058 0.40 6.5E−4 0.0040 0.45 6.4E−4 0.0040 0.58 Lower limit 1.65 1.38 0.639 1.88 1.46 0.615 1.92 1.46 0.565 of 95% CI Upper limit 8.78 6.86 3.10 10.3 7.31 2.98 11.2 7.31 2.76 of 95% CI OR Quartile 3 4.96 3.90 1.89 7.14 4.40 2.02 11.5 4.40 2.05 p Value 6.1E−4 0.0011 0.070 1.8E−4 5.6E−4 0.047 1.3E−4 5.6E−4 0.046 Lower limit 1.98 1.72 0.949 2.56 1.90 1.01 3.29 1.90 1.01 of 95% CI Upper limit 12.4 8.80 3.76 20.0 10.2 4.03 40.3 10.2 4.13 of 95% CI OR Quartile 4 2.87 2.71 2.88 3.75 2.59 3.07 5.23 2.59 2.94 p Value 0.066 0.057 0.013 0.039 0.069 0.010 0.030 0.069 0.017 Lower limit 0.935 0.970 1.25 1.07 0.927 1.30 1.18 0.927 1.21 of 95% CI Upper limit of 95% CI 8.80 7.56 6.65 13.2 7.25 7.25 23.3 7.25 7.12

TABLE 12.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or urine serum creatinine or output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 37700 57200 37400 58200 37400 58200 Average 42400 61000 41100 60600 40300 60400 Stdev 18900 26900 18500 26600 17100 26700 p (t-test) 1.3E−4 1.4E−4 1.2E−4 Min 20500 15600 20500 15600 20500 15600 Max 90400 141000 90400 141000 90400 141000 n (Patient) 38 106 33 111 31 113 sCr only Median 38000 58200 38000 58200 38000 58200 Average 44600 61300 44600 61300 44200 61200 Stdev 22600 26300 22600 26300 22000 26400 p (t-test) 3.1E−4 3.1E−4 3.2E−4 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 45 99 45 99 43 101 UO only Median 51300 59900 51300 58900 48300 59100 Average 52900 64300 53600 62500 51800 62900 Stdev 19000 30800 19300 30000 18700 29100 p (t-test) 0.015 0.062 0.021 Min 20500 15600 20500 15600 20500 15600 Max 97900 141000 97900 141000 97900 141000 n (Patient) 60 65 52 73 47 79 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.70 0.60 0.73 0.70 0.57 0.74 0.71 0.61 SE 0.045 0.044 0.051 0.045 0.044 0.052 0.045 0.044 0.051 p Value 8.7E−7 3.6E−6 0.058 1.6E−7 3.6E−6 0.17 6.1E−8 2.9E−6 0.039 nCohort Recovered 38 45 60 33 45 52 31 43 47 nCohort Non-recovered 106 99 65 111 99 73 113 101 79 Cutoff Quartile 2 36300 36300 37600 36300 36300 37600 36300 36300 37700 Sensitivity 81% 83% 77% 80% 83% 75% 80% 82% 76% Specificity 42% 42% 27% 42% 42% 25% 42% 42% 28% Cutoff Quartile 3 52700 52700 55100 52700 52700 55100 52700 52700 55100 Sensitivity 59% 61% 57% 59% 61% 55% 59% 60% 56% Specificity 76% 73% 57% 82% 73% 56% 84% 74% 60% Cutoff Quartile 4 70900 70900 74700 70900 70900 74700 70900 70900 74300 Sensitivity 30% 30% 32% 30% 30% 30% 30% 31% 32% Specificity 89% 87% 82% 91% 87% 81% 94% 88% 85% OR Quartile 2 3.13 3.52 1.21 2.98 3.52 1.02 2.83 3.32 1.21 p Value 0.0056 0.0018 0.64 0.010 0.0018 0.97 0.016 0.0030 0.65 Lower limit of 95% CI 1.40 1.60 0.538 1.30 1.60 0.447 1.21 1.50 0.531 Upper limit of 95% CI 7.01 7.76 2.73 6.86 7.76 2.32 6.60 7.33 2.75 OR Quartile 3 4.72 4.23 1.73 6.60 4.23 1.53 7.57 4.44 1.85 p Value 3.0E−4 2.6E−4 0.13 1.2E−4 2.6E−4 0.25 1.1E−4 2.3E−4 0.099 Lower limit of 95% CI 2.03 1.95 0.851 2.52 1.95 0.747 2.71 2.01 0.891 Upper limit of 95% CI 11.0 9.17 3.51 17.3 9.17 3.13 21.2 9.80 3.85 OR Quartile 4 3.68 2.83 2.13 4.23 2.83 1.81 6.24 3.37 2.65 p Value 0.022 0.034 0.077 0.024 0.034 0.17 0.016 0.020 0.041 Lower limit of 95% CI 1.20 1.08 0.922 1.21 1.08 0.773 1.41 1.21 1.04 Upper limit of 95% CI 11.2 7.38 4.90 14.8 7.38 4.25 27.6 9.37 6.72

TABLE 12.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 44300 62000 44100 62800 44100 62800 Average 47600 64300 47200 64500 47200 64500 Stdev 20900 29100 20600 29000 20600 29000 p (t-test) 7.7E−5 3.5E−5 3.5E−5 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 81 69 80 70 80 70 sCr only Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 42800 59300 41000 59300 41000 59300 Average 46100 62700 45600 62700 45600 62700 Stdev 20900 27900 20800 27700 20800 27700 p (t-test) 9.3E−5 5.1E−5 5.1E−5 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 67 83 65 85 65 85 UO only Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 47500 63200 47400 64500 47000 64500 Average 49000 65900 48500 66200 47900 66400 Stdev 20700 31400 20600 30900 20400 30500 p (t-test) 1.3E−4 4.8E−5 1.7E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 95 54 93 56 91 58 Recovery Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.68 0.66 0.69 0.69 0.67 0.69 0.69 0.69 SE 0.044 0.043 0.048 0.044 0.043 0.047 0.044 0.043 0.046 p Value 6.3E−5 2.2E−5 8.7E−4 1.9E−5 6.0E−6 2.1E−4 1.9E−5 6.0E−6 4.4E−5 nCohort Recovered 81 67 95 80 65 93 80 65 91 nCohort Non-recovered 69 83 54 70 85 56 70 85 58 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 80% 81% 78% 80% 81% 79% 80% 81% 79% Specificity 30% 33% 26% 30% 34% 27% 30% 34% 27% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 65% 63% 65% 66% 62% 66% 66% 62% 67% Specificity 63% 66% 58% 64% 66% 59% 64% 66% 60% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 38% 36% 41% 39% 36% 41% 39% 36% 41% Specificity 85% 88% 83% 86% 89% 84% 86% 89% 85% OR Quartile 2 1.65 2.05 1.25 1.71 2.21 1.35 1.71 2.21 1.45 p Value 0.19 0.060 0.58 0.16 0.038 0.46 0.16 0.038 0.35 Lower limit of 95% CI 0.777 0.970 0.569 0.805 1.04 0.614 0.805 1.04 0.663 Upper limit of 95% CI 3.52 4.32 2.75 3.65 4.66 2.96 3.65 4.66 3.18 OR Quartile 3 3.19 3.21 2.53 3.37 3.24 2.82 3.37 3.24 3.14 p Value 7.0E−4 6.8E−4 0.0084 3.9E−4 6.5E−4 0.0033 3.9E−4 6.5E−4 0.0012 Lower limit of 95% CI 1.63 1.64 1.27 1.72 1.65 1.41 1.72 1.65 1.57 Upper limit of 95% CI 6.23 6.29 5.06 6.60 6.36 5.62 6.60 6.36 6.26 OR Quartile 4 3.48 4.17 3.39 3.94 4.76 3.62 3.94 4.76 3.88 p Value 0.0018 0.0012 0.0017 7.6E−4 6.8E−4 0.0010 7.6E−4 6.8E−4 5.8E−4 Lower limit of 95% CI 1.59 1.76 1.58 1.77 1.93 1.68 1.77 1.93 1.79 Upper limit of 95% CI 7.61 9.90 7.29 8.75 11.7 7.81 8.75 11.7 8.41

Example 13. Use of Tenascin for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Tenascin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 13.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 36100 55200 35800 55200 36600 55100 Average 41800 58900 41400 58800 41800 58500 Stdev 22000 26200 22400 26100 22700 26100 p (t-test) 9.4E−4 0.0010 0.0018 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 32 118 30 120 29 121 sCr only Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 37100 55800 37100 55800 37400 55500 Average 43400 59700 43400 59700 43800 59400 Stdev 21400 26600 21400 26600 21600 26700 p (t-test) 6.0E−4 6.0E−4 0.0011 Min 20500 15600 20500 15600 20500 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 41 109 41 109 40 110 UO only Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 45700 58900 45700 58900 44700 58600 Average 47900 62700 47900 62300 47800 62200 Stdev 21700 28600 21900 28400 22100 28200 p (t-test) 5.1E−4 6.9E−4 7.1E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 76 73 74 75 73 76 Recovery Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.70 0.66 0.72 0.70 0.66 0.71 0.69 0.66 SE 0.046 0.045 0.045 0.047 0.045 0.045 0.048 0.046 0.044 p Value 3.8E−6 1.4E−5 4.3E−4 1.9E−6 1.4E−5 4.8E−4 7.9E−6 4.3E−5 4.3E−4 nCohort 32 41 76 30 41 74 29 40 73 Recovered nCohort 118 109 73 120 109 75 121 110 76 Non-recovered Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 80% 81% 81% 80% 81% 81% 79% 80% 82% Specificity 44% 41% 30% 47% 41% 31% 45% 40% 32% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 56% 57% 62% 56% 57% 61% 55% 56% 62% Specificity 72% 68% 61% 73% 68% 61% 72% 68% 62% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 30% 31% 33% 29% 31% 32% 29% 31% 32% Specificity 91% 90% 82% 90% 90% 81% 90% 90% 81% OR Quartile 2 3.05 2.97 1.83 3.50 2.97 1.96 3.12 2.67 2.04 p Value 0.0085 0.0064 0.12 0.0037 0.0064 0.082 0.0090 0.015 0.067 Lower limit 1.33 1.36 0.854 1.50 1.36 0.918 1.33 1.21 0.951 of 95% CI Upper limit 6.99 6.49 3.91 8.15 6.49 4.21 7.33 5.85 4.36 of 95% CI OR Quartile 3 3.24 2.84 2.46 3.48 2.84 2.46 3.26 2.68 2.60 p Value 0.0068 0.0070 0.0073 0.0058 0.0070 0.0074 0.0093 0.011 0.0045 Lower limit 1.38 1.33 1.28 1.43 1.33 1.27 1.34 1.25 1.35 of 95% CI Upper limit 7.60 6.07 4.76 8.43 6.07 4.76 7.93 5.74 5.04 of 95% CI OR Quartile 4 4.08 4.19 2.17 3.71 4.19 2.02 3.53 4.03 1.95 p Value 0.028 0.011 0.045 0.041 0.011 0.070 0.050 0.014 0.085 Lower limit 1.16 1.38 1.02 1.06 1.38 0.946 1.00 1.33 0.912 of 95% CI Upper limit 14.3 12.7 4.63 13.0 12.7 4.30 12.4 12.2 4.15 of 95% CI

TABLE 13.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 37100 58200 37400 56200 37400 56200 Average 42900 60600 43400 60000 43400 60000 Stdev 21400 26500 21800 26500 21800 26500 p (t-test) 1.2E−4 3.7E−4 3.7E−4 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 45 105 43 107 43 107 sCr only Recovery Period 24 48 72 Duration Non- Non- Non- Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 37300 57200 37400 56200 37400 56200 Average 43900 61000 44200 60700 44200 60700 Stdev 21800 26600 21900 26600 21900 26600 p (t-test) 1.3E−4 2.5E−4 2.5E−4 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 50 100 49 101 49 101 UO only Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 47000 62700 47000 62000 47000 62000 Average 48500 64500 48600 64000 48600 64000 Stdev 20800 29300 21000 29100 21000 29100 p (t-test) 1.7E−4 3.3E−4 3.3E−4 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 79 66 77 68 77 68 Recovery Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.70 0.67 0.70 0.70 0.66 0.70 0.70 0.66 SE 0.043 0.043 0.045 0.044 0.043 0.045 0.044 0.043 0.045 p Value 4.2E−7 1.7E−6 1.8E−4 3.6E−6 5.8E−6 3.2E−4 3.6E−6 5.8E−6 3.2E−4 nCohort 45 50 79 43 49 77 43 49 77 Recovered nCohort 105 100 66 107 101 68 107 101 68 Non-recovered Cutoff Quartile 2 34800 34800 35000 34800 34800 35000 34800 34800 35000 Sensitivity 81% 82% 82% 80% 81% 82% 80% 81% 82% Specificity 40% 40% 30% 40% 39% 31% 40% 39% 31% Cutoff Quartile 3 52000 52000 52200 52000 52000 52200 52000 52000 52200 Sensitivity 59% 59% 64% 58% 58% 63% 58% 58% 63% Specificity 71% 68% 61% 70% 67% 61% 70% 67% 61% Cutoff Quartile 4 70700 70700 70800 70700 70700 70800 70700 70700 70800 Sensitivity 31% 32% 36% 31% 32% 35% 31% 32% 35% Specificity 89% 88% 84% 88% 88% 83% 88% 88% 83% OR Quartile 2 2.83 3.04 1.96 2.68 2.73 2.11 2.68 2.73 2.11 p Value 0.0080 0.0043 0.093 0.013 0.0096 0.063 0.013 0.0096 0.063 Lower limit 1.31 1.42 0.893 1.23 1.28 0.961 1.23 1.28 0.961 of 95% CI Upper limit 6.12 6.51 4.32 5.81 5.85 4.65 5.81 5.85 4.65 of 95% CI OR Quartile 3 3.55 3.06 2.71 3.18 2.90 2.69 3.18 2.90 2.69 p Value 9.8E−4 0.0022 0.0038 0.0027 0.0036 0.0039 0.0027 0.0036 0.0039 Lower limit 1.67 1.50 1.38 1.49 1.42 1.37 1.49 1.42 1.37 of 95% CI Upper limit 7.54 6.25 5.32 6.77 5.93 5.28 6.77 5.93 5.28 of 95% CI OR Quartile 4 3.67 3.45 2.90 3.39 3.32 2.69 3.39 3.32 2.69 p Value 0.012 0.011 0.0073 0.019 0.013 0.013 0.019 0.013 0.013 Lower limit 1.33 1.33 1.33 1.22 1.28 1.24 1.22 1.28 1.24 of 95% CI Upper limit 10.1 8.93 6.32 9.39 8.61 5.84 9.39 8.61 5.84 of 95% CI

TABLE 13.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 41900 58900 41900 58900 41000 58600 Average 45400 61900 45400 61900 45300 61800 Stdev 21400 27100 21400 27100 21600 27000 p (t-test) 1.5E−4 1.5E−4 1.4E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 58 91 58 91 57 92 sCr only Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 42800 58900 42800 58900 42800 58900 Average 46100 62300 46100 62300 46100 62300 Stdev 21700 27300 21700 27300 21700 27300 p (t-test) 1.8E−4 1.8E−4 1.8E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 61 87 61 87 61 87 UO only Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 47800 62000 47800 62000 47600 62000 Average 51100 65300 51100 65300 50800 65100 Stdev 20500 30000 20500 30000 20800 29500 p (t-test) 0.0016 0.0016 0.0014 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 72 61 72 61 70 63 Recovery Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.68 0.64 0.69 0.68 0.64 0.69 0.68 0.64 SE 0.043 0.043 0.048 0.043 0.043 0.048 0.043 0.043 0.048 p Value 1.2E−5 2.7E−5 0.0041 1.2E−5 2.7E−5 0.0041 8.5E−6 2.7E−5 0.0027 nCohort 58 61 72 58 61 72 57 61 70 Recovered nCohort 91 87 61 91 87 61 92 87 63 Non-recovered Cutoff Quartile 2 35000 35000 37100 35000 35000 37100 35000 35000 37100 Sensitivity 81% 82% 79% 81% 82% 79% 82% 82% 79% Specificity 34% 34% 28% 34% 34% 28% 35% 34% 29% Cutoff Quartile 3 52200 52300 54300 52200 52300 54300 52200 52300 54300 Sensitivity 60% 60% 59% 60% 60% 59% 61% 60% 60% Specificity 66% 64% 57% 66% 64% 57% 67% 64% 59% Cutoff Quartile 4 70800 70900 71600 70800 70900 71600 70800 70900 71600 Sensitivity 34% 33% 38% 34% 33% 38% 34% 33% 37% Specificity 88% 87% 85% 88% 87% 85% 88% 87% 84% OR Quartile 2 2.29 2.33 1.42 2.29 2.33 1.42 2.38 2.33 1.54 p Value 0.032 0.029 0.39 0.032 0.029 0.39 0.024 0.029 0.29 Lower limit 1.08 1.09 0.638 1.08 1.09 0.638 1.12 1.09 0.691 of 95% CI Upper limit 4.88 4.97 3.16 4.88 4.97 3.16 5.08 4.97 3.43 of 95% CI OR Quartile 3 2.90 2.63 1.90 2.90 2.63 1.90 3.11 2.63 2.15 p Value 0.0023 0.0050 0.068 0.0023 0.0050 0.068 0.0013 0.0050 0.031 Lower limit 1.46 1.34 0.954 1.46 1.34 0.954 1.56 1.34 1.07 of 95% CI Upper limit 5.76 5.18 3.80 5.76 5.18 3.80 6.21 5.18 4.30 of 95% CI OR Quartile 4 3.76 3.31 3.36 3.76 3.31 3.36 3.63 3.31 3.08 p Value 0.0039 0.0068 0.0040 0.0039 0.0068 0.0040 0.0051 0.0068 0.0073 Lower limit 1.53 1.39 1.47 1.53 1.39 1.47 1.47 1.39 1.35 of 95% CI Upper limit 9.27 7.88 7.66 9.27 7.88 7.66 8.94 7.88 7.02 of 95% CI

TABLE 13.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 44300 58900 43700 59100 43500 58200 Average 47700 63000 46700 63200 46600 62700 Stdev 21600 27800 21500 27400 22000 27100 p (t-test) 3.9E−4 1.4E−4 2.3E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 65 79 62 82 59 85 sCr only Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 44300 58900 44300 58900 44100 58600 Average 47800 63400 47800 63400 47700 63200 Stdev 21500 28000 21500 28000 21600 27800 p (t-test) 3.0E−4 3.0E−4 3.2E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 67 77 67 77 66 78 UO only Recovery Period 24 48 72 Duration Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 51300 62000 48300 61000 50000 59400 Average 53100 66100 52700 65500 52900 64900 Stdev 20800 30700 21100 29800 21200 29500 p (t-test) 0.0057 0.0062 0.0097 Min 18800 15600 18800 15600 18800 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 70 55 65 60 64 62 Recovery Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.67 0.62 0.68 0.67 0.62 0.68 0.67 0.61 SE 0.045 0.045 0.051 0.044 0.045 0.050 0.044 0.045 0.050 p Value 2.1E−4 2.1E−4 0.020 2.6E−5 2.1E−4 0.014 3.7E−5 2.0E−4 0.022 nCohort Recovered 65 67 70 62 67 65 59 66 64 nCohort Non-recovered 79 77 55 82 77 60 85 78 62 Cutoff Quartile 2 36300 36300 37600 36300 36300 37600 36300 36300 37700 Sensitivity 82% 83% 80% 83% 83% 80% 84% 83% 81% Specificity 34% 34% 29% 35% 34% 29% 37% 35% 31% Cutoff Quartile 3 52700 52700 55100 52700 52700 55100 52700 52700 55100 Sensitivity 59% 60% 60% 61% 60% 58% 60% 60% 56% Specificity 62% 61% 57% 65% 61% 57% 64% 62% 56% Cutoff Quartile 4 70900 70900 74700 70900 70900 74700 70900 70900 74300 Sensitivity 34% 35% 35% 34% 35% 33% 33% 35% 32% Specificity 86% 87% 81% 87% 87% 82% 86% 86% 81% OR Quartile 2 2.38 2.57 1.60 2.67 2.57 1.65 3.02 2.67 1.89 p Value 0.028 0.018 0.27 0.013 0.018 0.23 0.0055 0.014 0.13 Lower limit of 95% CI 1.10 1.18 0.691 1.23 1.18 0.722 1.38 1.22 0.832 Upper limit of 95% CI 5.15 5.62 3.71 5.80 5.62 3.78 6.57 5.84 4.31 OR Quartile 3 2.35 2.34 2.00 2.84 2.34 1.85 2.71 2.49 1.67 p Value 0.013 0.013 0.058 0.0028 0.013 0.090 0.0044 0.0080 0.16 Lower limit of 95% CI 1.20 1.20 0.976 1.43 1.20 0.909 1.37 1.27 0.824 Upper limit of 95% CI 4.60 4.57 4.10 5.63 4.57 3.76 5.40 4.87 3.37 OR Quartile 4 3.23 3.48 2.31 3.50 3.48 2.21 3.13 3.35 2.06 p Value 0.0064 0.0038 0.045 0.0048 0.0038 0.060 0.010 0.0049 0.085 Lower limit of 95% CI 1.39 1.50 1.02 1.46 1.50 0.968 1.31 1.44 0.906 Upper limit of 95% CI 7.51 8.09 5.25 8.37 8.09 5.04 7.49 7.80 4.70

TABLE 13.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 46800 64300 45700 64500 44700 64600 Average 48100 65800 47900 65700 47500 66000 Stdev 20900 29800 20900 29500 20700 29400 p (t-test) 3.5E−5 2.7E−5 1.2E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 89 61 88 62 87 63 sCr only Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 44300 62000 44300 62000 44300 62000 Average 46700 64800 46700 64800 46700 64800 Stdev 20400 28800 20400 28800 20400 28800 p (t-test) 1.6E−5 1.6E−5 1.6E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 79 71 79 71 79 71 UO only Recovery 24 48 72 Period Non- Non- Non- Duration Recovered recovered Recovered recovered Recovered recovered (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 47400 64300 47400 64300 47200 64600 Average 48800 67300 48800 67300 48300 67500 Stdev 20800 31300 20800 31300 20700 30800 p (t-test) 3.2E−5 3.2E−5 1.1E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 98 51 98 51 96 53 Recovery Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.69 0.68 0.68 0.69 0.68 0.69 0.69 0.69 SE 0.045 0.043 0.048 0.045 0.043 0.048 0.044 0.043 0.047 p Value 6.9E−5 1.0E−5 2.2E−4 3.8E−5 1.0E−5 2.2E−4 1.2E−5 1.0E−5 4.4E−5 nCohort 89 79 98 88 79 98 87 79 96 Recovered nCohort 61 71 51 62 71 51 63 71 53 Non-recovered Cutoff 34800 34800 34700 34800 34800 34700 34800 34800 34700 Quartile 2 Sensitivity 80% 82% 80% 81% 82% 80% 81% 82% 81% Specificity 29% 32% 28% 30% 32% 28% 30% 32% 28% Cutoff 52000 52000 51800 52000 52000 51800 52000 52000 51800 Quartile 3 Sensitivity 64% 63% 67% 65% 63% 67% 65% 63% 68% Specificity 60% 62% 58% 60% 62% 58% 61% 62% 59% Cutoff 70700 70700 70500 70700 70700 70500 70700 70700 70500 Quartile 4 Sensitivity 41% 41% 41% 40% 41% 41% 41% 41% 42% Specificity 85% 89% 83% 85% 89% 83% 86% 89% 83% OR Quartile 2 1.69 2.07 1.56 1.75 2.07 1.56 1.81 2.07 1.68 p Value 0.19 0.063 0.29 0.16 0.063 0.29 0.13 0.063 0.21 Lower limit 0.773 0.960 0.686 0.802 0.960 0.686 0.832 0.960 0.742 of 95% CI Upper limit 3.67 4.44 3.54 3.81 4.44 3.54 3.95 4.44 3.82 of 95% CI OR Quartile 3 2.61 2.83 2.78 2.75 2.83 2.78 2.91 2.83 3.10 p Value 0.0052 0.0021 0.0046 0.0032 0.0021 0.0046 0.0019 0.0021 0.0017 Lower limit 1.33 1.46 1.37 1.40 1.46 1.37 1.48 1.46 1.53 of 95% CI Upper limit 5.11 5.49 5.64 5.40 5.49 5.64 5.70 5.49 6.27 of 95% CI OR Quartile 4 4.06 5.37 3.34 3.90 5.37 3.34 4.39 5.37 3.55 p Value 4.2E−4 8.8E−5 0.0020 6.0E−4 8.8E−5 0.0020 2.4E−4 8.8E−5 0.0012 Lower limit 1.86 2.32 1.55 1.79 2.32 1.55 1.99 2.32 1.65 of 95% CI Upper limit 8.85 12.4 7.16 8.48 12.4 7.16 9.67 12.4 7.63 of 95% CI

Example 14. Use of Follistatin-Related Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Follistatin-related protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 14.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 49500 81000 50300 81000 52000 77400 Average 81100 87400 82200 86200 81800 88000 Stdev 141000 43300 136000 42000 132000 43500 p (t-test) 0.75 0.84 0.77 Min 10500 20700 10500 20700 10500 20700 Max 1230000 204000 1230000 204000 1230000 204000 n (Patient) 95 55 103 47 110 40 sCr only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 49300 81400 50300 81000 50500 80900 Average 80700 88100 82800 84800 82300 86300 Stdev 142000 43200 137000 38700 134000 38800 p (t-test) 0.70 0.92 0.85 Min 10500 20700 10500 20700 10500 20700 Max 1230000 204000 1230000 162000 1230000 162000 n (Patient) 94 56 103 47 108 42 UO only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 60000 80700 60000 80700 60400 74700 Average 82700 90800 82700 90800 82900 89400 Stdev 123000 44800 123000 44800 122000 45600 p (t-test) 0.77 0.77 0.82 Min 10500 34800 10500 34800 10500 34800 Max 1230000 204000 1230000 204000 1230000 204000 n (Patient) 130 19 130 19 131 18 Persistence Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.68 0.65 0.66 0.65 0.65 0.65 0.66 0.63 SE 0.047 0.046 0.072 0.050 0.050 0.072 0.053 0.052 0.074 p Value 2.1E−4 7.8E−5 0.044 0.0017 0.0021 0.044 0.0033 0.0018 0.074 nCohort 95 94 130 103 103 130 110 108 131 Non-persistent nCohort 55 56 19 47 47 19 40 42 18 Persistent Cutoff 40800 40800 40400 40800 40800 40400 40800 40800 40400 Quartile 2 Sensitivity 85% 86% 89% 85% 85% 89% 88% 88% 89% Specificity 32% 32% 27% 30% 30% 27% 30% 31% 27% Cutoff 63600 63600 63900 63600 63600 63900 63600 63600 63900 Quartile 3 Sensitivity 69% 70% 68% 70% 70% 68% 70% 71% 67% Specificity 61% 62% 52% 59% 59% 52% 57% 58% 52% Cutoff 91200 91200 91300 91200 91200 91300 91200 91200 91300 Quartile 4 Sensitivity 40% 41% 37% 38% 38% 37% 38% 38% 33% Specificity 83% 84% 76% 81% 81% 76% 79% 80% 76% OR Quartile 2 2.71 2.81 3.13 2.46 2.46 3.13 3.00 3.26 2.92 p Value 0.024 0.019 0.14 0.052 0.052 0.14 0.035 0.023 0.17 Lower limit 1.14 1.18 0.688 0.994 0.994 0.688 1.08 1.17 0.638 of 95% CI Upper limit 6.44 6.68 14.3 6.09 6.09 14.3 8.34 9.03 13.3 of 95% CI OR Quartile 3 3.50 3.70 2.38 3.42 3.42 2.38 3.13 3.50 2.16 p Value 4.9E−4 2.8E−4 0.098 0.0011 0.0011 0.098 0.0039 0.0015 0.15 Lower limit 1.73 1.83 0.851 1.64 1.64 0.851 1.44 1.62 0.764 of 95% CI Upper limit 7.09 7.48 6.63 7.16 7.16 6.63 6.79 7.57 6.10 of 95% CI OR Quartile 4 3.29 3.67 1.86 2.58 2.58 1.86 2.27 2.41 1.55 p Value 0.0022 8.9E−4 0.23 0.015 0.015 0.23 0.041 0.027 0.42 Lower limit 1.54 1.70 0.675 1.20 1.20 0.675 1.03 1.10 0.537 of 95% CI Upper limit 7.05 7.90 5.14 5.53 5.53 5.14 4.99 5.24 4.46 of 95% CI

TABLE 14.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 48000 80900 48800 80900 49500 80700 Average 68600 104000 70400 105000 70400 110000 Stdev 77500 152000 76700 159000 74400 169000 p (t-test) 0.060 0.072 0.046 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 88 62 94 56 101 49 sCr only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 48000 80900 48400 80700 49100 80700 Average 81200 86600 82500 84900 81700 86900 Stdev 146000 42900 143000 40800 139000 41200 p (t-test) 0.78 0.90 0.80 Min 10500 20700 10500 20700 10500 20700 Max 1230000 204000 1230000 204000 1230000 204000 n (Patient) 88 62 93 57 99 51 UO only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 55100 81600 55100 81600 55200 80700 Average 72800 136000 72800 136000 73100 136000 Stdev 69600 228000 69600 228000 69400 233000 p (t-test) 0.011 0.011 0.012 Min 10500 34800 10500 34800 10500 34800 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 123 26 123 26 124 25 Persistence Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.68 0.68 0.67 0.67 0.68 0.68 0.67 0.67 SE 0.045 0.045 0.062 0.047 0.047 0.062 0.048 0.048 0.063 p Value 3.9E−5 5.8E−5 0.0029 1.9E−4 3.8E−4 0.0029 2.0E−4 2.9E−4 0.0058 nCohort 88 88 123 94 93 123 101 99 124 Non-persistent nCohort 62 62 26 56 57 26 49 51 25 Persistent Cutoff 40800 40800 40400 40800 40800 40400 40800 40800 40400 Quartile 2 Sensitivity 85% 85% 92% 86% 86% 92% 88% 88% 92% Specificity 33% 33% 28% 32% 32% 28% 32% 32% 28% Cutoff 63600 63600 63900 63600 63600 63900 63600 63600 63900 Quartile 3 Sensitivity 66% 66% 69% 68% 67% 69% 69% 69% 68% Specificity 61% 61% 54% 61% 60% 54% 59% 60% 53% Cutoff 91200 91200 91300 91200 91200 91300 91200 91200 91300 Quartile 4 Sensitivity 39% 39% 42% 38% 37% 42% 39% 37% 40% Specificity 84% 84% 78% 82% 82% 78% 81% 81% 77% OR Quartile 2 2.89 2.89 4.77 2.81 2.92 4.77 3.32 3.58 4.52 p Value 0.013 0.013 0.040 0.019 0.015 0.040 0.013 0.0085 0.048 Lower limit 1.26 1.26 1.07 1.18 1.23 1.07 1.28 1.38 1.01 of 95% CI Upper limit 6.67 6.67 21.3 6.68 6.92 21.3 8.61 9.26 20.2 of 95% CI OR Quartile 3 3.10 3.10 2.61 3.25 3.03 2.61 3.32 3.23 2.42 p Value 0.0011 0.0011 0.038 9.1E−4 0.0016 0.038 0.0012 0.0013 0.058 Lower limit 1.57 1.57 1.05 1.62 1.52 1.05 1.61 1.58 0.972 of 95% CI Upper limit 6.11 6.11 6.44 6.53 6.03 6.44 6.85 6.59 6.02 of 95% CI OR Quartile 4 3.34 3.34 2.61 2.72 2.61 2.61 2.73 2.50 2.29 p Value 0.0021 0.0021 0.034 0.0094 0.013 0.034 0.0096 0.018 0.073 Lower limit 1.55 1.55 1.07 1.28 1.23 1.07 1.28 1.17 0.925 of 95% CI Upper limit 7.18 7.18 6.33 5.78 5.53 6.33 5.85 5.33 5.65 of 95% CI

TABLE 14.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. sCr or UO Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 47100 74000 47300 77400 48200 73900 Average 68100 102000 68200 104000 69800 106000 Stdev 79600 146000 78800 149000 76600 59000 p (t-test) 0.069 0.060 0.060 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 83 67 86 64 94 56 sCr only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 46500 77400 47300 80900 47900 80700 Average 67400 103000 67600 105000 69100 107000 Stdev 79800 145000 78500 149000 76800 157000 p (t-test) 0.061 0.051 0.051 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 82 68 86 64 93 57 UO only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 53300 77200 53300 77200 53900 73800 Average 72300 122000 72300 122000 72700 123000 Stdev 71600 200000 71600 200000 71400 204000 p (t-test) 0.026 0.026 0.028 Min 10500 34800 10500 34800 10500 34800 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 115 34 115 34 116 33 Persistence Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.70 0.68 0.70 0.71 0.68 0.69 0.70 0.68 SE 0.044 0.043 0.055 0.044 0.044 0.055 0.046 0.045 0.056 p Value 7.5E−6 2.0E−6 8.3E−4 4.0E−6 1.6E−6 8.3E−4 3.6E−5 1.0E−5 0.0017 nCohort 83 82 115 86 86 115 94 93 116 Non-persistent nCohort 67 68 34 64 64 34 56 57 33 Persistent Cutoff 40800 40800 40400 40800 40800 40400 40800 40800 40400 Quartile 2 Sensitivity 87% 87% 94% 88% 88% 94% 89% 89% 94% Specificity 35% 35% 30% 35% 35% 30% 34% 34% 30% Cutoff 63600 63600 63900 63600 63600 63900 63600 63600 63900 Quartile 3 Sensitivity 67% 68% 71% 69% 69% 71% 70% 70% 70% Specificity 64% 65% 56% 64% 64% 56% 62% 62% 55% Cutoff 91200 91200 91300 91200 91200 91300 91200 91200 91300 Quartile 4 Sensitivity 37% 38% 35% 38% 39% 35% 36% 37% 33% Specificity 84% 85% 77% 84% 85% 77% 81% 82% 77% OR Quartile 2 3.46 3.59 7.00 3.75 3.75 7.00 4.30 4.46 6.70 p Value 0.0036 0.0027 0.010 0.0027 0.0027 0.010 0.0026 0.0020 0.012 Lower limit 1.50 1.56 1.59 1.58 1.58 1.59 1.67 1.73 1.52 of 95% CI Upper limit 7.97 8.27 30.8 8.89 8.89 30.8 11.1 11.5 29.5 of 95% CI OR Quartile 3 3.61 3.82 3.01 3.90 3.90 3.01 3.70 3.90 2.83 p Value 2.1E−4 1.1E−4 0.0088 1.0E−4 1.0E−4 0.0088 2.8E−4 1.6E−4 0.014 Lower limit 1.83 1.93 1.32 1.96 1.96 1.32 1.83 1.93 1.24 of 95% CI Upper limit 7.12 7.55 6.87 7.77 7.77 6.87 7.48 7.90 6.48 of 95% CI OR Quartile 4 3.21 3.61 1.87 3.09 3.60 1.87 2.35 2.61 1.65 p Value 0.0031 0.0013 0.14 0.0038 0.0012 0.14 0.026 0.013 0.24 Lower limit 1.48 1.65 0.816 1.44 1.66 0.816 1.11 1.23 0.710 of 95% CI Upper limit 6.93 7.91 4.27 6.63 7.81 4.27 4.97 5.53 3.83 of 95% CI

TABLE 14.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 47100 74000 47500 74000 48400 73800 Average 67800 102000 68500 103000 70300 104000 Stdev 80400 144000 79200 148000 77700 155000 p (t-test) 0.072 0.069 0.083 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 81 69 85 65 91 59 sCr only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 47100 74000 47300 77400 48200 73900 Average 67700 102000 67800 103000 69700 104000 Stdev 80300 144000 79400 147000 78000 154000 p (t-test) 0.069 0.061 0.073 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 81 69 84 66 90 60 UO only Persistence 24 48 72 Period Non- Non- Non- Duration persistent Persistent persistent Persistent persistent Persistent (hr) Cohort Cohort Cohort Cohort Cohort Cohort Median 52900 77200 53100 73800 53300 72700 Average 71300 118000 71700 118000 72100 118000 Stdev 72600 186000 72400 188000 72200 190000 p (t-test) 0.029 0.032 0.035 Min 10500 34800 10500 34800 10500 34800 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 109 40 110 39 111 38 Persistence Period Duration 24 48 72 (hr) sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.70 0.69 0.70 0.70 0.68 0.68 0.69 0.67 SE 0.043 0.043 0.052 0.044 0.043 0.053 0.046 0.045 0.053 p Value 5.2E−6 4.3E−6 2.9E−4 8.5E−6 2.3E−6 6.2E−4 1.1E−4 3.9E−5 0.0013 nCohort 81 81 109 85 84 110 91 90 111 Non-persistent nCohort 69 69 40 65 66 39 59 60 38 Persistent Cutoff 40800 40800 40400 40800 40800 40400 40800 40800 40400 Quartile 2 Sensitivity 87% 87% 92% 88% 88% 92% 88% 88% 92% Specificity 36% 36% 31% 35% 36% 31% 34% 34% 31% Cutoff 63600 63600 63900 63600 63600 63900 63600 63600 63900 Quartile 3 Sensitivity 67% 67% 70% 68% 68% 69% 68% 68% 68% Specificity 64% 64% 57% 64% 64% 56% 62% 62% 56% Cutoff 91200 91200 91300 91200 91200 91300 91200 91200 91300 Quartile 4 Sensitivity 38% 38% 38% 37% 38% 36% 34% 35% 34% Specificity 85% 85% 79% 84% 85% 78% 80% 81% 77% OR Quartile 2 3.72 3.72 5.59 3.89 4.03 5.37 3.84 3.98 5.15 p Value 0.0021 0.0021 0.0067 0.0021 0.0016 0.0082 0.0034 0.0026 0.0099 Lower limit 1.61 1.61 1.61 1.64 1.70 1.55 1.56 1.62 1.48 of 95% CI Upper limit 8.57 8.57 19.4 9.22 9.55 18.6 9.44 9.79 17.9 of 95% CI OR Quartile 3 3.59 3.59 3.08 3.65 3.86 2.91 3.37 3.55 2.74 p Value 2.1E−4 2.1E−4 0.0045 2.0E−4 1.1E−4 0.0072 5.6E−4 3.2E−4 0.011 Lower limit 1.82 1.82 1.42 1.85 1.95 1.34 1.69 1.78 1.26 of 95% CI Upper limit 7.05 7.05 6.68 7.22 7.64 6.32 6.72 7.09 5.98 of 95% CI OR Quartile 4 3.48 3.48 2.24 2.97 3.33 2.01 2.08 2.31 1.79 p Value 0.0018 0.0018 0.045 0.0052 0.0023 0.086 0.054 0.028 0.16 Lower limit 1.59 1.59 1.02 1.38 1.54 0.906 0.986 1.09 0.800 of 95% CI Upper limit 7.61 7.61 4.94 6.37 7.21 4.45 4.39 4.89 4.00 of 95% CI

TABLE 14.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 45600 73900 45900 74000 47700 73900 Average 67300 101000 67400 102000 69300 103000 Stdev 81600 141000 80700 144000 78700 151000 p (t-test) 0.073 0.065 0.074 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 78 72 81 69 88 62 sCr only Median 45900 73800 46500 73900 47700 72700 Average 67900 101000 68000 102000 69900 103000 Stdev 81300 142000 80400 145000 78800 151000 p (t-test) 0.081 0.072 0.086 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 79 71 82 68 88 62 UO only Median 51800 80700 51800 80700 52900 77200 Average 70400 119000 70400 119000 70900 119000 Stdev 72400 183000 72400 183000 72200 186000 p (t-test) 0.022 0.022 0.024 Min 10500 34800 10500 34800 10500 34800 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 108 41 108 41 109 40 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.70 0.70 0.70 0.71 0.70 0.70 0.69 0.68 0.69 SE 0.043 0.043 0.051 0.043 0.043 0.051 0.045 0.045 0.052 p Value 1.5E−6 5.0E−6 8.2E−5 8.5E−7 2.8E−6 8.2E−5 1.9E−5 4.8E−5 1.9E−4 nCohort 78 79 108 81 82 108 88 88 109 Non-persistent nCohort Persistent 72 71 41 69 68 41 62 62 40 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 88% 87% 93% 88% 88% 93% 89% 89% 92% Specificity 37% 37% 31% 37% 37% 31% 35% 35% 31% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 67% 66% 71% 68% 68% 71% 68% 68% 70% Specificity 65% 65% 57% 65% 65% 57% 62% 62% 57% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 38% 37% 39% 38% 37% 39% 35% 34% 38% Specificity 86% 85% 80% 85% 84% 80% 82% 81% 79% OR Quartile 2 4.14 4.00 5.82 4.49 4.33 5.82 4.27 4.27 5.59 p Value 8.6E−4 0.0012 0.0055 6.6E−4 8.9E−4 0.0055 0.0016 0.0016 0.0067 Lower limit of 1.80 1.73 1.68 1.89 1.82 1.68 1.74 1.74 1.61 95% CI Upper limit of 9.56 9.21 20.2 10.6 10.3 20.2 10.5 10.5 19.4 95% CI OR Quartile 3 3.78 3.57 3.26 4.04 3.82 3.26 3.50 3.50 3.08 p Value 1.2E−4 2.2E−4 0.0028 6.0E−5 1.1E−4 0.0028 3.4E−4 3.4E−4 0.0045 Lower limit of 1.92 1.82 1.50 2.04 1.93 1.50 1.76 1.76 1.42 95% CI Upper limit of 7.43 7.00 7.06 8.00 7.55 7.06 6.95 6.95 6.68 95% CI OR Quartile 4 3.65 3.23 2.50 3.48 3.09 2.50 2.48 2.14 2.24 p Value 0.0014 0.0033 0.022 0.0018 0.0042 0.022 0.018 0.046 0.045 Lower limit of 1.65 1.48 1.14 1.59 1.43 1.14 1.17 1.01 1.02 95% CI Upper limit of 8.10 7.05 5.47 7.61 6.67 5.47 5.25 4.51 4.94 95% CI

Example 15. Use of Follistatin-Related Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Follistatin-related protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 15.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 41100 71900 45900 71700 47900 74000 Average 52000 99200 84100 82800 82300 84900 Stdev 40900 136000 158000 41000 149000 41400 p (t-test) 0.018 0.95 0.89 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 1230000 204000 1230000 204000 n (Patient) 50 100 75 75 85 65 sCr only Median 41800 71900 45400 74000 48500 80900 Average 64000 93800 66800 99300 69700 102000 Stdev 96900 123000 83700 137000 79800 149000 p (t-test) 0.13 0.084 0.089 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 52 98 73 77 86 64 UO only Median 54800 82000 55600 80700 55800 77200 Average 77300 103000 83200 85900 82700 89000 Stdev 118000 107000 127000 41900 125000 43000 p (t-test) 0.24 0.91 0.81 Min 10500 24400 10500 24400 10500 34000 Max 1230000 689000 1230000 204000 1230000 204000 n (Patient) 112 37 120 29 125 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.77 0.76 0.66 0.67 0.71 0.63 0.68 0.68 0.64 SE 0.038 0.039 0.054 0.044 0.042 0.061 0.045 0.045 0.065 p Value 9.5E−13 3.3E−11 0.0029 7.4E−5 7.4E−7 0.036 8.9E−5 3.8E−5 0.027 nCohort 50 52 112 75 73 120 85 86 125 Non-persistent nCohort Persistent 100 98 37 75 77 29 65 64 24 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 87% 87% 89% 85% 86% 86% 86% 86% 88% Specificity 50% 48% 29% 36% 37% 28% 34% 34% 27% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 65% 65% 70% 65% 68% 69% 68% 69% 71% Specificity 80% 79% 56% 65% 68% 54% 64% 64% 54% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 33% 33% 38% 35% 38% 31% 35% 38% 33% Specificity 90% 88% 79% 84% 88% 76% 82% 84% 76% OR Quartile 2 6.69 6.05 3.45 3.27 3.52 2.37 3.22 3.11 2.62 p Value 3.6E−6 9.7E−6 0.030 0.0035 0.0019 0.13 0.0060 0.0077 0.14 Lower limit of 2.99 2.73 1.13 1.48 1.59 0.767 1.40 1.35 0.733 95% CI Upper limit of 15.0 13.4 10.5 7.25 7.80 7.33 7.42 7.16 9.33 95% CI OR Quartile 3 7.43 7.02 3.04 3.55 4.52 2.63 3.65 3.90 2.81 p Value 1.1E−6 1.1E−6 0.0063 2.2E−4 1.7E−5 0.029 2.0E−4 1.0E−4 0.033 Lower limit of 3.32 3.20 1.37 1.81 2.28 1.11 1.85 1.96 1.09 95% CI Upper limit of 16.6 15.4 6.75 6.96 8.98 6.24 7.22 7.77 7.24 95% CI OR Quartile 4 4.43 3.72 2.23 2.79 4.30 1.41 2.56 3.09 1.58 p Value 0.0040 0.0067 0.050 0.0100 6.3E−4 0.45 0.015 0.0038 0.34 Lower limit of 1.61 1.44 1.000 1.28 1.86 0.579 1.20 1.44 0.617 95% CI Upper limit of 12.2 9.61 4.98 6.07 9.91 3.44 5.44 6.63 4.06 95% CI

TABLE 15.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 40400 72000 45000 73900 45900 81000 Average 50500 99400 66400 98400 66400 102000 Stdev 39900 135000 85200 135000 81100 142000 p (t-test) 0.014 0.090 0.056 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 689000 1230000 689000 1230000 n (Patient) 49 101 70 80 79 71 sCr only Median 41800 72000 45000 73900 45900 80700 Average 62700 94100 66200 98500 67500 101000 Stdev 97500 122000 85100 135000 81500 142000 p (t-test) 0.11 0.087 0.074 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 51 99 70 80 79 71 UO only Median 50300 85100 52900 82200 53100 82400 Average 64600 127000 71900 118000 71800 125000 Stdev 41700 193000 72400 190000 71100 203000 p (t-test) 0.0022 0.032 0.018 Min 10500 24400 10500 24400 10500 34000 Max 265000 1230000 689000 1230000 689000 1230000 n (Patient) 103 46 111 38 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.79 0.77 0.71 0.71 0.72 0.68 0.72 0.71 0.69 SE 0.037 0.038 0.048 0.042 0.041 0.053 0.042 0.043 0.056 p Value 1.0E−14 7.6E−13 1.9E−5 2.5E−7 1.9E−7 7.7E−4 1.7E−7 1.2E−6 4.7E−4 nCohort 49 51 103 70 70 111 79 79 116 Non-persistent nCohort Persistent 101 99 46 80 80 38 71 71 33 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 87% 87% 91% 86% 86% 89% 87% 87% 91% Specificity 51% 49% 32% 39% 39% 30% 37% 37% 29% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 65% 66% 72% 66% 66% 71% 69% 68% 73% Specificity 82% 80% 59% 69% 69% 57% 67% 66% 56% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 34% 33% 41% 38% 38% 37% 39% 38% 39% Specificity 92% 90% 82% 89% 89% 78% 87% 86% 78% OR Quartile 2 7.05 6.36 4.95 3.94 3.94 3.60 4.00 4.00 4.15 p Value 2.2E−6 6.0E−6 0.0046 7.6E−4 7.6E−4 0.024 0.0012 0.0012 0.026 Lower limit of 3.14 2.86 1.64 1.77 1.77 1.18 1.73 1.73 1.19 95% CI Upper limit of 15.8 14.2 15.0 8.75 8.75 10.9 9.21 9.21 14.5 95% CI OR Quartile 3 8.38 7.84 3.69 4.28 4.28 3.22 4.54 4.02 3.40 p Value 5.4E−7 5.6E−7 6.8E−4 3.2E−5 3.2E−5 0.0039 1.6E−5 6.2E−5 0.0048 Lower limit of 3.65 3.50 1.74 2.16 2.16 1.45 2.28 2.04 1.45 95% CI Upper limit of 19.2 17.6 7.83 8.50 8.50 7.14 9.03 7.94 7.95 95% CI OR Quartile 4 5.71 4.60 3.11 4.65 4.65 2.11 4.49 3.79 2.37 p Value 0.0020 0.0032 0.0038 4.9E−4 4.9E−4 0.066 3.1E−4 0.0010 0.041 Lower limit of 1.90 1.67 1.44 1.96 1.96 0.951 1.99 1.71 1.04 95% CI Upper limit of 17.2 12.7 6.72 11.0 11.0 4.70 10.2 8.42 5.41 95% CI

TABLE 15.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 40400 72000 44700 73800 45200 74000 Average 50500 99400 66500 97800 66600 100000 Stdev 39900 135000 85900 134000 83200 139000 p (t-test) 0.014 0.097 0.074 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 689000 1230000 689000 1230000 n (Patient) 49 101 69 81 75 75 sCr only Median 41800 72000 44700 73800 45200 74000 Average 62700 94100 66400 98000 66500 100000 Stdev 97500 122000 85800 134000 83100 139000 p (t-test) 0.11 0.094 0.072 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 51 99 69 81 75 75 UO only Median 50100 82000 52900 77200 53100 73800 Average 63900 122000 71900 112000 71800 117000 Stdev 41800 184000 73900 178000 72600 188000 p (t-test) 0.0034 0.053 0.034 Min 10500 24400 10500 24400 10500 34000 Max 265000 1230000 689000 1230000 689000 1230000 n (Patient) 98 51 105 44 110 39 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.79 0.77 0.71 0.71 0.71 0.67 0.72 0.72 0.68 SE 0.037 0.038 0.047 0.042 0.041 0.051 0.042 0.041 0.052 p Value 1.0E−14 7.6E−13 9.0E−6 2.9E−7 2.3E−7 7.8E−4 1.2E−7 9.1E−8 5.7E−4 nCohort 49 51 98 69 69 105 75 75 110 Non-persistent nCohort Persistent 101 99 51 81 81 44 75 75 39 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 87% 87% 92% 86% 86% 91% 88% 88% 92% Specificity 51% 49% 34% 39% 39% 31% 39% 39% 31% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 65% 66% 71% 65% 65% 68% 68% 68% 69% Specificity 82% 80% 60% 68% 68% 57% 68% 68% 56% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 34% 33% 39% 37% 37% 34% 37% 37% 36% Specificity 92% 90% 82% 88% 88% 78% 87% 87% 78% OR Quartile 2 7.05 6.36 5.97 4.09 4.09 4.58 4.62 4.62 5.37 p Value 2.2E−6 6.0E−6 0.0015 5.5E−4 5.5E−4 0.0070 3.4E−4 3.4E−4 0.0082 Lower limit of 3.14 2.86 1.98 1.84 1.84 1.51 2.00 2.00 1.55 95% CI Upper limit of 15.8 14.2 18.0 9.09 9.09 13.9 10.7 10.7 18.6 95% CI OR Quartile 3 8.38 7.84 3.63 4.04 4.04 2.86 4.52 4.52 2.91 p Value 5.4E−7 5.6E−7 5.0E−4 6.0E−5 6.0E−5 0.0056 1.7E−5 1.7E−5 0.0072 Lower limit of 3.65 3.50 1.76 2.04 2.04 1.36 2.27 2.27 1.34 95% CI Upper limit of 19.2 17.6 7.50 8.00 8.00 6.01 8.97 8.97 6.32 95% CI OR Quartile 4 5.71 4.60 2.87 4.49 4.49 1.84 3.87 3.87 2.01 p Value 0.0020 0.0032 0.0066 6.6E−4 6.6E−4 0.12 0.0011 0.0011 0.086 Lower limit of 1.90 1.67 1.34 1.89 1.89 0.849 1.72 1.72 0.906 95% CI Upper limit of 17.2 12.7 6.13 10.6 10.6 4.01 8.74 8.74 4.45 95% CI

TABLE 15.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 40400 72000 45000 72700 45300 73900 Average 50500 99400 66900 97200 66900 99500 Stdev 39900 135000 86400 133000 83700 138000 p (t-test) 0.014 0.11 0.083 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 689000 1230000 689000 1230000 n (Patient) 49 101 68 82 74 76 sCr only Median 41800 72000 45000 72700 45300 73900 Average 62700 94100 66700 97300 66800 99600 Stdev 97500 122000 86400 133000 83700 138000 p (t-test) 0.11 0.11 0.081 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 51 99 68 82 74 76 UO only Median 50300 81200 51800 73800 52900 72700 Average 64200 118000 71900 108000 71900 112000 Stdev 42300 179000 75500 169000 74000 178000 p (t-test) 0.0059 0.075 0.053 Min 10500 24400 10500 24400 10500 34000 Max 265000 1230000 689000 1230000 689000 1230000 n (Patient) 95 54 100 49 105 44 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.79 0.77 0.69 0.71 0.71 0.66 0.71 0.72 0.67 SE 0.037 0.038 0.046 0.042 0.042 0.049 0.042 0.042 0.051 p Value 1.0E−14 7.6E−13 3.3E−5 6.3E−7 5.0E−7 0.0010 2.7E−7 2.1E−7 8.3E−4 nCohort 49 51 95 68 68 100 74 74 105 Non-persistent nCohort Persistent 101 99 54 82 82 49 76 76 44 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 87% 87% 91% 87% 87% 90% 88% 88% 91% Specificity 51% 49% 34% 40% 40% 32% 39% 39% 31% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 65% 66% 69% 65% 65% 67% 67% 67% 68% Specificity 82% 80% 60% 68% 68% 58% 68% 68% 57% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 34% 33% 37% 37% 37% 33% 37% 37% 34% Specificity 92% 90% 81% 88% 88% 78% 86% 86% 78% OR Quartile 2 7.05 6.36 4.98 4.25 4.25 4.14 4.80 4.80 4.58 p Value 2.2E−6 6.0E−6 0.0019 3.9E−4 3.9E−4 0.0061 2.4E−4 2.4E−4 0.0070 Lower limit of 3.14 2.86 1.81 1.91 1.91 1.50 2.08 2.08 1.51 95% CI Upper limit of 15.8 14.2 13.7 9.46 9.46 11.4 11.1 11.1 13.9 95% CI OR Quartile 3 8.38 7.84 3.26 3.82 3.82 2.85 4.25 4.25 2.86 p Value 5.4E−7 5.6E−7 0.0010 1.1E−4 1.1E−4 0.0042 3.3E−5 3.3E−5 0.0056 Lower limit of 3.65 3.50 1.61 1.93 1.93 1.39 2.15 2.15 1.36 95% CI Upper limit of 19.2 17.6 6.61 7.55 7.55 5.83 8.41 8.41 6.01 95% CI OR Quartile 4 5.71 4.60 2.52 4.33 4.33 1.72 3.73 3.73 1.84 p Value 0.0020 0.0032 0.016 8.9E−4 8.9E−4 0.16 0.0015 0.0015 0.12 Lower limit of 1.90 1.67 1.18 1.82 1.82 0.802 1.66 1.66 0.849 95% CI Upper limit of 17.2 12.7 5.35 10.3 10.3 3.68 8.42 8.42 4.01 95% CI

TABLE 15.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 40400 72000 43900 73800 44300 77400 Average 49300 99000 56600 104000 56800 107000 Stdev 39000 134000 40100 146000 40300 150000 p (t-test) 0.014 0.012 0.0076 Min 10500 20700 10500 20700 10500 20700 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 47 103 65 85 70 80 sCr only Median 41800 71900 45000 71300 45300 72700 Average 63400 93500 67500 96000 67500 98200 Stdev 98400 122000 87500 132000 84700 136000 p (t-test) 0.13 0.13 0.10 Min 10500 20700 10500 20700 10500 20700 Max 689000 1230000 689000 1230000 689000 1230000 n (Patient) 50 100 66 84 72 78 UO only Median 49500 81800 49800 81200 50300 77200 Average 62900 117000 64000 118000 64600 122000 Stdev 41600 175000 42100 179000 41900 186000 p (t-test) 0.0049 0.0054 0.0041 Min 10500 24400 10500 24400 10500 34000 Max 265000 1230000 265000 1230000 265000 1230000 n (Patient) 92 57 95 54 99 50 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.79 0.76 0.71 0.73 0.70 0.70 0.74 0.70 0.70 SE 0.036 0.039 0.045 0.040 0.042 0.046 0.040 0.042 0.047 p Value 1.6E−15 1.0E−11 4.3E−6 9.7E−9 2.5E−6 2.3E−5 7.3E−10 1.2E−6 3.7E−5 nCohort 47 50 92 65 66 95 70 72 99 Non-persistent nCohort Persistent 103 100 57 85 84 54 80 78 50 Cutoff Quartile 2 40800 40800 40400 40800 40800 40400 40800 40800 40400 Sensitivity 86% 86% 91% 87% 86% 91% 89% 87% 92% Specificity 51% 48% 35% 42% 39% 34% 41% 39% 33% Cutoff Quartile 3 63600 63600 63900 63600 63600 63900 63600 63600 63900 Sensitivity 65% 65% 68% 65% 63% 69% 68% 65% 68% Specificity 83% 80% 61% 69% 67% 60% 70% 67% 59% Cutoff Quartile 4 91200 91200 91300 91200 91200 91300 91200 91200 91300 Sensitivity 34% 33% 39% 38% 36% 37% 39% 36% 38% Specificity 94% 90% 83% 91% 88% 81% 90% 86% 81% OR Quartile 2 6.63 5.67 5.55 4.78 3.90 4.98 5.58 4.33 5.75 p Value 3.9E−6 1.7E−5 9.2E−4 1.3E−4 6.9E−4 0.0019 6.1E−5 4.3E−4 0.0019 Lower limit of 2.97 2.57 2.01 2.14 1.78 1.81 2.41 1.91 1.91 95% CI Upper limit of 14.8 12.5 15.3 10.7 8.56 13.7 12.9 9.78 17.3 95% CI OR Quartile 3 9.07 7.43 3.37 4.12 3.42 3.26 4.85 3.78 3.01 p Value 5.3E−7 1.1E−6 6.5E−4 5.6E−5 3.7E−4 0.0010 8.1E−6 1.2E−4 0.0026 Lower limit of 3.83 3.32 1.68 2.07 1.74 1.61 2.42 1.92 1.47 95% CI Upper limit of 21.5 16.6 6.77 8.22 6.73 6.61 9.69 7.43 6.15 95% CI OR Quartile 4 7.55 4.43 2.99 5.94 4.03 2.52 5.69 3.47 2.58 p Value 0.0014 0.0040 0.0047 2.3E−4 0.0016 0.016 1.5E−4 0.0027 0.014 Lower limit of 2.19 1.61 1.40 2.30 1.70 1.18 2.31 1.54 1.21 95% CI Upper limit of 26.0 12.2 6.37 15.3 9.55 5.35 14.0 7.82 5.51 95% CI

Example 16. Use of Basigin for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Basigin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 16.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 8720 16900 8860 16900 9030 15600 Average 13300 20300 14200 19400 14500 19500 Stdev 12900 14700 13800 13800 13500 14800 p (t-test) 0.0027 0.034 0.053 Min 1970 3580 1970 3580 1970 3580 Max 72100 69500 72100 69500 72100 69500 n (Patient) 95 55 103 47 110 40 sCr only Median 8730 16800 8930 16700 9030 14300 Average 13300 20100 14200 19500 14500 19300 Stdev 12900 14600 13600 14200 13400 14900 p (t-test) 0.0038 0.030 0.060 Min 1970 3580 1970 3580 1970 3580 Max 72100 69500 72100 69500 72100 69500 n (Patient) 94 56 103 47 108 42 UO only Median 9370 18800 9370 18800 9380 18300 Average 14900 22800 14900 22800 14900 23000 Stdev 13600 15100 13600 15100 13500 15500 p (t-test) 0.020 0.020 0.021 Min 1970 5570 1970 5570 1970 5570 Max 72100 69500 72100 69500 72100 69500 n (Patient) 130 19 130 19 131 18 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.71 0.70 0.71 0.68 0.67 0.71 0.65 0.65 0.71 SE 0.046 0.046 0.070 0.049 0.049 0.070 0.053 0.052 0.072 p Value 6.5E−6 1.2E−5 0.0026 2.0E−4 3.9E−4 0.0026 0.0035 0.0050 0.0043 nCohort 95 94 130 103 103 130 110 108 131 Non-persistent nCohort Persistent 55 56 19 47 47 19 40 42 18 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 91% 91% 95% 91% 91% 95% 90% 90% 94% Specificity 35% 35% 28% 33% 33% 28% 31% 31% 27% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 75% 73% 84% 74% 72% 84% 72% 69% 83% Specificity 64% 64% 55% 61% 60% 55% 58% 57% 54% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 36% 36% 42% 34% 34% 42% 30% 29% 44% Specificity 81% 81% 77% 79% 79% 77% 76% 76% 77% OR Quartile 2 5.32 5.52 6.89 5.30 5.30 6.89 4.03 4.36 6.44 p Value 0.0012 9.3E−4 0.065 0.0031 0.0031 0.065 0.014 0.0091 0.075 Lower limit of 1.94 2.01 0.887 1.76 1.76 0.887 1.33 1.44 0.827 95% CI Upper limit of 14.6 15.2 53.6 16.0 16.0 53.6 12.2 13.2 50.2 95% CI OR Quartile 3 5.25 4.82 6.42 4.59 3.95 6.42 3.67 3.01 5.92 p Value 1.0E−5 2.1E−5 0.0044 9.6E−5 3.3E−4 0.0044 0.0013 0.0044 0.0068 Lower limit of 2.51 2.33 1.78 2.14 1.87 1.78 1.66 1.41 1.63 95% CI Upper limit of 11.0 9.97 23.1 9.88 8.38 23.1 8.09 6.41 21.4 95% CI OR Quartile 4 2.44 2.35 2.42 1.90 1.90 2.42 1.38 1.26 2.69 p Value 0.020 0.026 0.082 0.10 0.10 0.082 0.43 0.57 0.056 Lower limit of 1.15 1.11 0.894 0.884 0.884 0.894 0.618 0.566 0.976 95% CI Upper limit of 5.18 4.97 6.58 4.09 4.09 6.58 3.10 2.81 7.43 95% CI

TABLE 16.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 8330 16200 8730 16800 8930 16700 Average 12600 20500 13100 20500 13500 20700 Stdev 11600 15700 12400 15300 12100 16200 p (t-test) 4.6E−4 0.0016 0.0027 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 88 62 94 56 101 49 sCr only Median 8580 15100 8740 15700 8930 14100 Average 12800 20200 13400 19900 13800 19900 Stdev 11700 15700 12600 15200 12300 16000 p (t-test) 0.0013 0.0047 0.011 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 88 62 93 57 99 51 UO only Median 9260 17500 9260 17500 9310 17200 Average 14700 21500 14700 21500 14800 21600 Stdev 13700 14300 13700 14300 13600 14600 p (t-test) 0.024 0.024 0.026 Min 1970 4850 1970 4850 1970 4850 Max 72100 69500 72100 69500 72100 69500 n (Patient) 123 26 123 26 124 25 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.71 0.70 0.70 0.71 0.69 0.70 0.68 0.66 0.69 SE 0.044 0.044 0.061 0.045 0.046 0.061 0.048 0.048 0.063 p Value 1.1E−6 7.7E−6 0.0015 4.7E−6 2.1E−5 0.0015 2.0E−4 7.7E−4 0.0025 nCohort 88 88 123 94 93 123 101 99 124 Non-persistent nCohort Persistent 62 62 26 56 57 26 49 51 25 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 89% 89% 92% 89% 89% 92% 88% 88% 92% Specificity 35% 35% 28% 34% 34% 28% 32% 32% 28% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 74% 73% 81% 75% 74% 81% 73% 71% 80% Specificity 67% 66% 56% 65% 65% 56% 61% 61% 56% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 35% 34% 38% 36% 33% 38% 33% 29% 40% Specificity 82% 81% 77% 81% 80% 77% 78% 77% 77% OR Quartile 2 4.27 4.27 4.77 4.30 4.46 4.77 3.32 3.58 4.52 p Value 0.0016 0.0016 0.040 0.0026 0.0020 0.040 0.013 0.0085 0.048 Lower limit of 1.74 1.74 1.07 1.67 1.73 1.07 1.28 1.38 1.01 95% CI Upper limit of 10.5 10.5 21.3 11.1 11.5 21.3 8.61 9.26 20.2 95% CI OR Quartile 3 5.85 5.12 5.37 5.55 5.09 5.37 4.40 3.69 5.02 p Value 1.6E−6 6.8E−6 0.0015 5.4E−6 1.1E−5 0.0015 1.1E−4 4.1E−4 0.0024 Lower limit of 2.84 2.51 1.90 2.65 2.46 1.90 2.08 1.79 1.77 95% CI Upper limit of 12.0 10.4 15.2 11.6 10.5 15.2 9.32 7.62 14.2 95% CI OR Quartile 4 2.48 2.14 2.12 2.35 1.95 2.12 1.74 1.38 2.29 p Value 0.018 0.046 0.100 0.026 0.080 0.100 0.15 0.41 0.073 Lower limit of 1.17 1.01 0.866 1.11 0.923 0.866 0.813 0.643 0.925 95% CI Upper limit of 5.25 4.51 5.19 4.97 4.11 5.19 3.73 2.95 5.65 95% CI

TABLE 16.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 8000 15700 8050 16200 8730 16800 Average 11900 20800 11900 21100 12300 21900 Stdev 11000 15600 11000 15700 10700 16600 p (t-test) 6.4E−5 4.5E−5 3.1E−5 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 83 67 86 64 94 56 sCr only Median 8010 15100 8160 15100 8740 14500 Average 11900 20600 12300 20700 12600 21100 Stdev 11100 15600 11300 15700 11000 16500 p (t-test) 9.9E−5 2.0E−4 2.2E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 82 68 86 64 93 57 UO only Median 9000 18300 9000 18300 9030 17700 Average 13400 24500 13400 24500 13400 24700 Stdev 11700 17400 11700 17400 11700 17600 p (t-test) 2.7E−5 2.7E−5 2.8E−5 Min 1970 4850 1970 4850 1970 4850 Max 67300 72100 67300 72100 67300 72100 n (Patient) 115 34 115 34 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.74 0.73 0.74 0.74 0.73 0.74 0.73 0.71 0.74 SE 0.041 0.042 0.053 0.042 0.042 0.053 0.044 0.045 0.053 p Value 7.7E−9 1.6E−8 4.5E−6 3.9E−9 6.4E−8 4.5E−6 3.4E−7 4.2E−6 9.4E−6 nCohort 83 82 115 86 86 115 94 93 116 Non-persistent nCohort Persistent 67 68 34 64 64 34 56 57 33 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 90% 90% 94% 91% 91% 94% 89% 89% 94% Specificity 37% 38% 30% 37% 37% 30% 34% 34% 30% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 76% 75% 82% 78% 77% 82% 77% 74% 82% Specificity 71% 71% 59% 71% 70% 59% 66% 65% 59% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 36% 35% 44% 36% 34% 44% 36% 33% 45% Specificity 83% 83% 80% 83% 81% 80% 81% 80% 80% OR Quartile 2 5.11 5.30 7.00 5.73 5.73 7.00 4.30 4.46 6.70 p Value 3.8E−4 2.9E−4 0.010 3.1E−4 3.1E−4 0.010 0.0026 0.0020 0.012 Lower limit of 2.08 2.15 1.59 2.22 2.22 1.59 1.67 1.73 1.52 95% CI Upper limit of 12.6 13.0 30.8 14.8 14.8 30.8 11.1 11.5 29.5 95% CI OR Quartile 3 7.84 7.25 6.75 8.71 7.54 6.75 6.41 5.09 6.38 p Value 4.1E−8 9.0E−8 9.2E−5 1.8E−8 8.5E−8 9.2E−5 1.3E−6 1.1E−5 1.5E−4 Lower limit of 3.76 3.51 2.59 4.10 3.60 2.59 3.02 2.46 2.44 95% CI Upper limit of 16.3 15.0 17.6 18.5 15.8 17.6 13.6 10.5 16.6 95% CI OR Quartile 4 2.75 2.65 3.16 2.66 2.29 3.16 2.35 1.95 3.37 p Value 0.0092 0.012 0.0058 0.011 0.030 0.0058 0.026 0.080 0.0038 Lower limit of 1.28 1.24 1.40 1.25 1.08 1.40 1.11 0.923 1.48 95% CI Upper limit of 5.89 5.67 7.15 5.65 4.85 7.15 4.97 4.11 7.68 95% CI

TABLE 16.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 7920 16700 8000 16700 8440 16900 Average 11400 21000 11900 21100 12100 21600 Stdev 10700 15500 11100 15600 10800 16200 p (t-test) 1.6E−5 4.0E−5 3.3E−5 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 81 69 85 65 91 59 sCr only Median 7920 15700 8010 16200 8580 16800 Average 11800 20600 11900 20900 12200 21400 Stdev 11100 15400 11100 15600 10900 16200 p (t-test) 8.7E−5 6.3E−5 5.5E−5 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 81 69 84 66 90 60 UO only Median 8930 18400 8940 18000 8950 17900 Average 12600 24800 12900 24500 12900 24600 Stdev 10500 18000 10700 18100 10700 18300 p (t-test) 1.0E−6 4.3E−6 4.5E−6 Min 1970 4850 1970 4850 1970 4850 Max 60400 72100 60400 72100 60400 72100 n (Patient) 109 40 110 39 111 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.76 0.74 0.75 0.75 0.75 0.74 0.73 0.73 0.74 SE 0.040 0.041 0.049 0.041 0.041 0.050 0.043 0.043 0.050 p Value 1.9E−10 4.8E−9 2.2E−7 1.1E−9 2.6E−9 1.2E−6 5.9E−8 1.3E−7 2.8E−6 nCohort 81 81 109 85 84 110 91 90 111 Non-persistent nCohort Persistent 69 69 40 65 66 39 59 60 38 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 90% 90% 92% 91% 91% 92% 90% 90% 92% Specificity 38% 38% 31% 38% 38% 31% 35% 36% 31% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 77% 75% 82% 78% 77% 82% 76% 75% 82% Specificity 73% 72% 61% 72% 71% 61% 67% 67% 60% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 36% 35% 42% 35% 35% 41% 36% 35% 42% Specificity 84% 83% 81% 82% 82% 80% 81% 81% 80% OR Quartile 2 5.49 5.49 5.59 5.94 6.15 5.37 4.79 4.97 5.15 p Value 2.1E−4 2.1E−4 0.0067 2.3E−4 1.7E−4 0.0082 0.0012 9.2E−4 0.0099 Lower limit of 2.23 2.23 1.61 2.30 2.39 1.55 1.86 1.93 1.48 95% CI Upper limit of 13.5 13.5 19.4 15.3 15.9 18.6 12.4 12.8 17.9 95% CI OR Quartile 3 8.88 7.71 7.52 9.26 8.50 7.12 6.54 6.00 6.74 p Value 8.4E−9 4.2E−8 1.2E−5 8.2E−9 1.8E−8 2.0E−5 7.1E−7 1.5E−6 3.5E−5 Lower limit of 4.23 3.72 3.05 4.34 4.03 2.89 3.11 2.89 2.73 95% CI Upper limit of 18.7 16.0 18.5 19.7 17.9 17.6 13.7 12.5 16.7 95% CI OR Quartile 4 2.97 2.55 3.10 2.56 2.46 2.78 2.41 2.31 2.94 p Value 0.0056 0.016 0.0049 0.015 0.019 0.011 0.022 0.028 0.0078 Lower limit of 1.38 1.19 1.41 1.20 1.16 1.26 1.14 1.09 1.33 95% CI Upper limit of 6.42 5.46 6.81 5.44 5.23 6.14 5.09 4.89 6.52 95% CI

TABLE 16.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 7630 15100 7700 15700 8330 16800 Average 11500 20500 11700 20800 11900 21400 Stdev 10900 15400 10900 15500 10600 16100 p (t-test) 5.7E−5 4.2E−5 2.5E−5 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 78 72 81 69 88 62 sCr only Median 7700 14500 7810 15100 8330 15600 Average 11900 20300 12000 20500 12300 21000 Stdev 11200 15300 11200 15500 11000 16100 p (t-test) 1.8E−4 1.4E−4 1.3E−4 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 79 71 82 68 88 62 UO only Median 8900 18000 8900 18000 8930 17900 Average 12600 24600 12600 24600 12700 24700 Stdev 10600 17900 10600 17900 10500 18100 p (t-test) 1.5E−6 1.5E−6 1.6E−6 Min 1970 4850 1970 4850 1970 4850 Max 60400 72100 60400 72100 60400 72100 n (Patient) 108 41 108 41 109 40 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.75 0.73 0.75 0.75 0.74 0.75 0.74 0.72 0.75 SE 0.040 0.041 0.048 0.040 0.041 0.048 0.043 0.043 0.049 p Value 1.2E−9 1.2E−8 1.9E−7 6.8E−10 6.9E−9 1.9E−7 3.1E−8 3.1E−7 4.5E−7 nCohort 78 79 108 81 82 108 88 88 109 Non-persistent nCohort Persistent 72 71 41 69 68 41 62 62 40 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 90% 90% 93% 91% 91% 93% 90% 90% 92% Specificity 40% 39% 31% 40% 39% 31% 36% 36% 31% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 74% 73% 83% 75% 75% 83% 74% 73% 82% Specificity 72% 71% 62% 72% 71% 62% 67% 66% 61% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 35% 34% 41% 35% 34% 41% 35% 34% 42% Specificity 83% 82% 81% 83% 82% 81% 82% 81% 81% OR Quartile 2 6.12 5.90 5.82 6.86 6.61 5.82 5.33 5.33 5.59 p Value 8.2E−5 1.1E−4 0.0055 6.9E−5 9.4E−5 0.0055 5.3E−4 5.3E−4 0.0067 Lower limit of 2.49 2.40 1.68 2.66 2.56 1.68 2.07 2.07 1.61 95% CI Upper limit of 15.1 14.5 20.2 17.7 17.1 20.2 13.8 13.8 19.4 95% CI OR Quartile 3 7.10 6.66 7.94 7.71 7.25 7.94 5.85 5.12 7.52 p Value 9.4E−8 2.0E−7 6.7E−6 4.2E−8 9.0E−8 6.7E−6 1.6E−6 6.8E−6 1.2E−5 Lower limit of 3.46 3.26 3.22 3.72 3.51 3.22 2.84 2.51 3.05 95% CI Upper limit of 14.6 13.6 19.6 16.0 15.0 19.6 12.0 10.4 18.5 95% CI OR Quartile 4 2.66 2.37 2.93 2.55 2.28 2.93 2.48 2.14 3.10 p Value 0.013 0.026 0.0070 0.016 0.031 0.0070 0.018 0.046 0.0049 Lower limit of 1.23 1.11 1.34 1.19 1.08 1.34 1.17 1.01 1.41 95% CI Upper limit of 5.73 5.06 6.42 5.46 4.84 6.42 5.25 4.51 6.81 95% CI

Example 17. Use of Basigin for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Basigin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 17.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5980 14300 7700 14500 8440 16700 Average 6980 20300 11700 20000 12100 20700 Stdev 4140 15000 11300 15100 10900 16000 p (t-test) 6.6E−9 2.1E−4 1.3E−4 Min 1970 3580 1970 3580 1970 3580 Max 25700 72100 60400 72100 60400 72100 n (Patient) 50 100 75 75 85 65 sCr only Median 6250 14100 7530 14500 8580 15000 Average 8250 19900 11300 20100 12800 19900 Stdev 7670 14800 11200 15000 12200 15200 p (t-test) 4.3E−7 8.3E−5 0.0019 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 60400 72100 60400 72100 n (Patient) 52 98 73 77 86 64 UO only Median 9030 18000 9190 19600 9370 21700 Average 14200 21200 14400 22300 14500 23100 Stdev 13700 13600 13600 14100 13400 15000 p (t-test) 0.0077 0.0054 0.0052 Min 1970 3580 1970 3580 1970 3580 Max 72100 69500 72100 69500 72100 69500 n (Patient) 112 37 120 29 125 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.87 0.84 0.70 0.73 0.75 0.71 0.71 0.70 0.71 SE 0.028 0.032 0.053 0.041 0.039 0.058 0.043 0.044 0.063 p Value 0 0 1.7E−4 1.2E−8 1.7E−10 3.4E−4 1.9E−6 9.5E−6 0.0011 nCohort 50 52 112 75 73 120 85 86 125 Non-persistent nCohort Persistent 100 98 37 75 77 29 65 64 24 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 91% 91% 92% 88% 90% 90% 86% 88% 92% Specificity 58% 56% 30% 39% 41% 28% 34% 35% 28% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 69% 68% 78% 71% 71% 79% 71% 70% 79% Specificity 88% 85% 59% 71% 73% 57% 66% 65% 55% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 37% 36% 43% 36% 36% 48% 38% 36% 50% Specificity 98% 94% 80% 85% 86% 80% 85% 83% 79% OR Quartile 2 14.0 12.5 4.94 4.62 6.02 3.43 3.22 3.75 4.28 p Value 5.4E−9 1.7E−8 0.012 3.4E−4 5.1E−5 0.055 0.0060 0.0027 0.057 Lower limit of 5.76 5.19 1.42 2.00 2.53 0.973 1.40 1.58 0.955 95% CI Upper limit of 33.9 30.0 17.2 10.7 14.3 12.1 7.42 8.89 19.2 95% CI OR Quartile 3 16.3 11.9 5.20 5.80 6.62 5.01 4.68 4.42 4.68 p Value 9.1E−9 2.1E−8 2.0E−4 9.4E−7 2.1E−7 0.0011 1.5E−5 2.8E−5 0.0038 Lower limit of 6.30 5.00 2.18 2.87 3.25 1.90 2.33 2.20 1.64 95% CI Upper limit of 42.3 28.2 12.4 11.7 13.5 13.2 9.39 8.87 13.3 95% CI OR Quartile 4 28.8 9.07 3.12 3.27 3.60 3.73 3.46 2.66 3.81 p Value 0.0011 4.7E−4 0.0054 0.0035 0.0020 0.0025 0.0017 0.011 0.0040 Lower limit of 3.81 2.63 1.40 1.48 1.60 1.59 1.60 1.25 1.53 95% CI Upper limit of 217 31.3 6.94 7.25 8.12 8.78 7.50 5.65 9.45 95% CI

TABLE 17.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5750 14100 7520 15000 8000 16700 Average 6860 20200 10900 20200 11400 20800 Stdev 4090 14900 10900 14900 10600 15500 p (t-test) 7.0E−9 3.3E−5 2.1E−5 Min 1970 3580 1970 3580 1970 3580 Max 25700 72100 60400 72100 60400 72100 n (Patient) 49 101 70 80 79 71 sCr only Median 6200 14100 7520 14300 8090 15600 Average 8160 19800 11400 19800 12400 19800 Stdev 7720 14800 11400 14800 12300 14700 p (t-test) 4.7E−7 1.7E−4 0.0010 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 60400 72100 60400 72100 n (Patient) 51 99 70 80 79 71 UO only Median 8860 18400 8950 21700 9030 24500 Average 12500 23500 12900 24800 13100 25800 Stdev 11200 16500 11300 17200 11200 18000 p (t-test) 5.4E−6 2.4E−6 1.9E−6 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 103 46 111 38 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.88 0.84 0.74 0.77 0.75 0.75 0.74 0.72 0.75 SE 0.028 0.031 0.046 0.038 0.039 0.050 0.041 0.042 0.053 p Value 0 0 1.6E−7 4.5E−12 2.1E−10 4.8E−7 3.5E−9 3.0E−7 2.6E−6 nCohort 49 51 103 70 70 111 79 79 116 Non-persistent nCohort Persistent 101 99 46 80 80 38 71 71 33 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 91% 91% 91% 89% 89% 89% 87% 87% 91% Specificity 59% 57% 32% 41% 41% 30% 37% 37% 29% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 69% 69% 80% 72% 71% 82% 73% 72% 82% Specificity 90% 86% 63% 76% 74% 60% 71% 70% 59% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 37% 35% 46% 36% 35% 50% 38% 35% 52% Specificity 98% 94% 83% 87% 86% 83% 86% 84% 82% OR Quartile 2 14.8 13.2 4.95 5.58 5.58 3.60 4.00 4.00 4.15 p Value 3.0E−9 9.7E−9 0.0046 6.1E−5 6.1E−5 0.024 0.0012 0.0012 0.026 Lower limit of 6.08 5.46 1.64 2.41 2.41 1.18 1.73 1.73 1.19 95% CI Upper limit of 36.1 31.8 15.0 12.9 12.9 10.9 9.21 9.21 14.5 95% CI OR Quartile 3 19.9 13.8 7.03 8.22 7.16 6.74 6.66 5.84 6.38 p Value 8.4E−9 1.3E−8 4.2E−6 1.9E−8 9.2E−8 3.5E−5 2.0E−7 9.3E−7 1.5E−4 Lower limit of 7.19 5.59 3.06 3.94 3.48 2.73 3.26 2.89 2.44 95% CI Upper limit of 54.9 34.0 16.1 17.1 14.7 16.7 13.6 11.8 16.6 95% CI OR Quartile 4 27.8 8.75 4.25 3.85 3.23 4.84 3.79 2.76 4.81 p Value 0.0013 5.9E−4 2.7E-4 0.0015 0.0046 1.2E−4 0.0010 0.0097 2.1E−4 Lower limit of 3.68 2.54 1.95 1.67 1.43 2.16 1.71 1.28 2.10 95% CI Upper limit of 95% CI 209 30.1 9.26 8.88 7.28 10.8 8.42 5.95 11.0

TABLE 17.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5750 14100 7500 14500 7700 15600 Average 6860 20200 10900 20000 10800 20400 Stdev 4090 14900 11000 14900 10600 15200 p (t-test) 7.0E−9 4.1E−5 4.4E−5 Min 1970 3580 1970 3580 1970 3580 Max 25700 72100 60400 72100 60400 72100 n (Patient) 49 101 69 81 75 75 sCr only Median 6200 14100 7500 14100 7700 14500 Average 8160 19800 11400 19700 11700 20000 Stdev 7720 14800 11500 14800 11300 15200 p (t-test) 4.7E−7 2.2E−4 2.1E−4 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 60400 72100 60400 72100 n (Patient) 51 99 69 81 75 75 UO only Median 8730 18000 8860 19200 8940 18800 Average 11700 24100 12100 25000 12400 25800 Stdev 9830 17000 10000 17600 10100 18000 p (t-test) 8.3E−8 9.4E−8 8.0E−8 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 98 51 105 44 110 39 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.88 0.84 0.77 0.77 0.75 0.76 0.75 0.73 0.76 SE 0.028 0.031 0.043 0.038 0.039 0.046 0.040 0.041 0.049 p Value 0 0 5.1E−10 4.2E−12 2.1E−10 1.5E−8 4.6E−10 1.4E−8 1.3E−7 nCohort 49 51 98 69 69 105 75 75 110 Non-persistent nCohort Persistent 101 99 51 81 81 44 75 75 39 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 91% 91% 92% 89% 89% 91% 88% 88% 92% Specificity 59% 57% 34% 42% 42% 31% 39% 39% 31% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 69% 69% 82% 72% 70% 82% 73% 72% 82% Specificity 90% 86% 66% 75% 74% 63% 73% 72% 61% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 37% 35% 45% 36% 35% 48% 36% 35% 49% Specificity 98% 94% 85% 87% 86% 84% 85% 84% 83% OR Quartile 2 14.8 13.2 5.97 5.80 5.80 4.58 4.62 4.62 5.37 p Value 3.0E−9 9.7E−9 0.0015 4.3E−5 4.3E−5 0.0070 3.4E−4 3.4E−4 0.0082 Lower limit of 6.08 5.46 1.98 2.50 2.50 1.51 2.00 2.00 1.55 95% CI Upper limit of 36.1 31.8 18.0 13.5 13.5 13.9 10.7 10.7 18.6 95% CI OR Quartile 3 19.9 13.8 9.19 7.71 6.73 7.62 7.56 6.61 7.12 p Value 8.4E−9 1.3E−8 1.8E−7 4.2E−8 2.0E−7 3.9E−6 4.3E−8 2.1E−7 2.0E−5 Lower limit of 7.19 5.59 4.00 3.72 3.28 3.22 3.67 3.24 2.89 95% CI Upper limit of 54.9 34.0 21.1 16.0 13.8 18.0 15.6 13.5 17.6 95% CI OR Quartile 4 27.8 8.75 4.55 3.72 3.12 4.73 3.27 2.79 4.55 p Value 0.0013 5.9E−4 1.4E-4 0.0021 0.0061 1.1E−4 0.0035 0.0100 2.0E−4 Lower limit of 3.68 2.54 2.09 1.61 1.38 2.15 1.48 1.28 2.05 95% CI Upper limit of 95% CI 209 30.1 9.90 8.57 7.02 10.4 7.25 6.07 10.1

TABLE 17.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5750 14100 7300 15000 7630 15700 Average 6860 20200 10800 20000 11200 20400 Stdev 4090 14900 11000 14800 10900 15100 p (t-test) 7.0E−9 3.7E−5 3.7E−5 Min 1970 3580 1970 3580 1970 3580 Max 25700 72100 60400 72100 60400 72100 n (Patient) 49 101 68 82 74 76 sCr only Median 6200 14100 7330 14300 7630 15000 Average 8160 19800 11300 19700 11600 20000 Stdev 7720 14800 11500 14700 11300 15100 p (t-test) 4.7E−7 1.9E−4 1.8E−4 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 60400 72100 60400 72100 n (Patient) 51 99 68 82 74 76 UO only Median 8720 17900 8800 18000 8860 17900 Average 11700 23300 12100 23700 12400 24300 Stdev 9930 16800 10200 17200 10200 17800 p (t-test) 3.7E−7 8.1E−7 9.1E−7 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 95 54 100 49 105 44 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.88 0.84 0.76 0.77 0.76 0.75 0.75 0.74 0.74 SE 0.028 0.031 0.043 0.038 0.039 0.045 0.039 0.040 0.047 p Value 0 0 2.5E−9  5.4E−13  3.6E−11 3.6E−8 9.8E−11 2.9E−9 3.8E−7 nCohort 49 51 95 68 68 100 74 74 105 Non-persistent nCohort Persistent 101 99 54 82 82 49 76 76 44 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 91% 91% 91% 89% 89% 90% 88% 88% 91% Specificity 59% 57% 34% 43% 43% 32% 39% 39% 31% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 69% 69% 80% 72% 71% 80% 74% 72% 80% Specificity 90% 86% 66% 76% 75% 64% 74% 73% 62% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 37% 35% 43% 35% 34% 43% 36% 34% 43% Specificity 98% 94% 84% 87% 85% 83% 85% 84% 82% OR Quartile 2 14.8 13.2 4.98 6.03 6.03 4.14 4.80 4.80 4.58 p Value 3.0E−9 9.7E−9 0.0019 2.9E−5 2.9E−5 0.0061 2.4E−4 2.4E−4 0.0070 Lower limit of 6.08 5.46 1.81 2.60 2.60 1.50 2.08 2.08 1.51 95% CI Upper limit of 36.1 31.8 13.7 14.0 14.0 11.4 11.1 11.1 13.9 95% CI OR Quartile 3 19.9 13.8 7.70 8.34 7.25 6.93 8.11 7.07 6.32 p Value 8.4E−9 1.3E−8 3.7E−7 1.9E−8 9.0E−8 2.5E−6 1.9E−8 9.4E−8 1.4E−5 Lower limit of 7.19 5.59 3.50 3.98 3.51 3.10 3.91 3.45 2.75 95% CI Upper limit of 54.9 34.0 16.9 17.5 15.0 15.5 16.8 14.5 14.5 95% CI OR Quartile 4 27.8 8.75 3.96 3.59 3.01 3.66 3.16 2.69 3.44 p Value 0.0013 5.9E−4 4.7E−4 0.0027 0.0078 9.5E−4 0.0046 0.013 0.0018 Lower limit of 3.68 2.54 1.83 1.56 1.34 1.70 1.43 1.23 1.58 95% CI Upper limit of 209 30.1 8.56 8.27 6.77 7.91 6.98 5.85 7.48 95% CI

TABLE 17.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 5750 14100 7010 14500 7520 15700 Average 6870 20000 10400 20000 10700 20400 Stdev 4170 14900 10500 14800 10400 15100 p (t-test) 2.3E−8 1.8E−5 1.2E−5 Min 1970 3580 1970 3580 1970 3580 Max 25700 72100 60400 72100 60400 72100 n (Patient) 47 103 65 85 70 80 sCr only Median 6250 14000 7330 14000 7630 14300 Average 8220 19700 11400 19400 11700 19600 Stdev 7790 14800 11700 14600 11500 15000 p (t-test) 8.9E−7 4.2E−4 4.3E−4 Min 1970 3580 1970 3580 1970 3580 Max 42400 72100 60400 72100 60400 72100 n (Patient) 50 100 66 84 72 78 UO only Median 8350 17700 8440 18400 8720 18400 Average 11400 23100 11400 23800 11700 24300 Stdev 9790 16600 9650 16800 9670 17200 p (t-test) 2.6E−7 6.4E−8 5.1E−8 Min 1970 3580 1970 3580 1970 3580 Max 60400 72100 60400 72100 60400 72100 n (Patient) 92 57 95 54 99 50 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCR or UO sCr only UO only AUC 0.87 0.84 0.76 0.78 0.75 0.77 0.77 0.73 0.77 SE 0.028 0.032 0.042 0.037 0.039 0.042 0.038 0.041 0.044 p Value 0 0 3.9E−10 3.3E−14 4.0E−10 1.7E−10 1.7E−12 2.3E−8 8.7E−10 nCohort 47 50 92 65 66 95 70 72 99 Non-persistent nCohort Persistent 103 100 57 85 84 54 80 78 50 Cutoff Quartile 2 6820 6820 6810 6820 6820 6810 6820 6820 6810 Sensitivity 90% 90% 91% 89% 88% 91% Specificity 60% 56% 35% 45% 42% 34% 89% 87% 92% Cutoff Quartile 3 11000 11000 11000 11000 11000 11000 11000 11000 11000 Sensitivity 68% 68% 79% 71% 69% 80% 72% 71% 80% Specificity 89% 86% 67% 77% 74% 66% 76% 72% 65% Cutoff Quartile 4 19900 19900 20100 19900 19900 20100 19900 19900 20100 Sensitivity 36% 35% 42% 35% 33% 44% 36% 33% 46% Specificity 98% 94% 85% 88% 85% 85% 87% 83% 85% OR Quartile 2 13.7 11.5 5.55 6.80 5.45 4.98 5.58 4.33 5.75 p Value 4.4E−9 2.7E−8 9.2E−4 9.0E−6 5.2E−5 0.0019 6.1E−5 4.3E−4 0.0019 Lower limit of 5.72 4.85 2.01 2.92 2.40 1.81 2.41 1.91 1.91 95% CI Upper limit of 32.9 27.1 15.3 15.9 12.4 13.7 12.9 9.78 17.3 95% CI OR Quartile 3 17.8 13.1 7.75 8.00 6.43 7.70 8.22 6.22 7.31 p Value 2.7E−8 2.4E−8 2.0E−7 4.0E−8 4.1E−7 3.7E−7 1.9E−8 4.4E−7 1.3E−6 Lower limit of 6.45 5.29 3.58 3.81 3.13 3.50 3.94 3.06 3.27 95% CI Upper limit of 49.2 32.2 16.8 16.8 13.2 16.9 17.1 12.6 16.4 95% CI OR Quartile 4 25.8 8.44 4.05 3.89 2.80 4.63 3.85 2.50 4.77 p Value 0.0016 7.3E−4 4.0E−4 0.0021 0.013 1.2E−4 0.0015 0.021 9.0E−5 Lower limit of 3.42 2.45 1.87 1.64 1.24 2.12 1.67 1.15 2.18 95% CI Upper limit of 195 29.1 8.79 9.22 6.30 10.1 8.88 5.44 10.4 95% CI

Example 18. Use of Cathepsin B for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Cathepsin B is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 18.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.42 8.44 2.42 8.44 2.83 7.78 Average 6.51 22.2 7.06 23.8 8.21 23.5 Stdev 10.3 29.0 10.9 30.7 14.2 30.1 p (t-test) 3.8E−6 2.6E−6 5.9E−5 Min 0.0435 0.240 0.0435 0.366 0.0435 0.366 Max 57.5 111 57.5 111 103 111 n (Patient) 97 51 105 43 112 36 sCr only Median 2.46 8.29 2.51 8.44 3.04 6.80 Average 6.57 21.8 6.84 24.3 8.05 23.1 Stdev 10.4 28.8 10.4 30.8 13.9 29.8 p (t-test) 7.2E−6 7.9E−7 5.4E−5 Min 0.0435 0.172 0.0435 0.172 0.0435 0.172 Max 57.5 111 57.5 111 103 111 n (Patient) 96 52 105 43 110 38 UO only Median 3.62 6.68 3.61 8.62 3.61 8.62 Average 10.2 25.1 10.1 26.4 10.1 26.4 Stdev 17.9 30.5 17.9 30.8 17.9 30.8 p (t-test) 0.0032 0.0016 0.0016 Min 0.0435 0.592 0.0435 0.592 0.0435 0.592 Max 111 91.6 111 91.6 111 91.6 n (Patient) 129 18 130 17 130 17 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.68 0.64 0.71 0.70 0.66 0.68 0.66 0.66 SE 0.047 0.047 0.074 0.049 0.050 0.076 0.054 0.054 0.076 p Value 2.9E−5 1.4E−4 0.052 2.4E−5 9.8E−5 0.038 8.1E−4 0.0029 0.038 nCohort Non-persistent 97 96 129 105 105 130 112 110 130 nCohort Persistent 51 52 18 43 43 17 36 38 17 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 86% 85% 83% 88% 86% 82% 86% 84% 82% Specificity 31% 30% 26% 30% 30% 26% 29% 28% 26% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 67% 65% 56% 70% 67% 59% 67% 63% 59% Specificity 59% 58% 50% 58% 57% 51% 55% 55% 51% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 43% 42% 44% 42% 44% 47% 44% 45% 47% Specificity 85% 84% 78% 82% 83% 78% 81% 82% 78% OR Quartile 2 2.81 2.38 1.79 3.33 2.58 1.65 2.48 2.09 1.65 p Value 0.025 0.051 0.38 0.021 0.052 0.45 0.084 0.13 0.45 Lower limit of 95% CI 1.14 0.997 0.488 1.20 0.990 0.447 0.886 0.797 0.447 Upper limit of 95% CI 6.97 5.68 6.57 9.25 6.74 6.11 6.94 5.50 6.11 OR Quartile 3 2.85 2.64 1.27 3.20 2.76 1.47 2.48 2.06 1.47 p Value 0.0038 0.0065 0.64 0.0026 0.0076 0.46 0.024 0.062 0.46 Lower limit of 95% CI 1.40 1.31 0.471 1.50 1.31 0.528 1.13 0.963 0.528 Upper limit of 95% CI 5.79 5.33 3.42 6.82 5.82 4.11 5.45 4.39 4.11 OR Quartile 4 4.15 3.96 2.76 3.26 3.83 3.10 3.47 3.64 3.10 p Value 3.6E−4 5.3E−4 0.051 0.0031 8.4E−4 0.033 0.0027 0.0016 0.033 Lower limit of 95% CI 1.90 1.82 0.997 1.49 1.74 1.10 1.54 1.63 1.10 Upper limit of 95% CI 9.06 8.63 7.63 7.13 8.41 8.74 7.80 8.13 8.74

TABLE 18.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.30 8.29 2.37 8.44 2.57 7.54 Average 5.93 21.2 6.29 22.6 7.68 21.9 Stdev 9.88 27.6 10.1 28.9 13.9 28.2 p (t-test) 4.0E−6 1.4E−6 6.5E−5 Min 0.0435 0.240 0.0435 0.366 0.0435 0.366 Max 57.5 111 57.5 111 103 111 n (Patient) 90 58 97 51 104 44 sCr only Median 2.40 7.97 2.40 7.97 2.83 6.80 Average 6.34 20.6 6.66 21.6 8.10 20.4 Stdev 10.4 27.7 10.6 28.8 14.3 27.8 p (t-test) 1.9E−5 1.0E−5 5.3E−4 Min 0.0435 0.172 0.0435 0.172 0.0435 0.172 Max 57.5 111 57.5 111 103 111 n (Patient) 90 58 96 52 102 46 UO only Median 3.63 4.73 3.62 6.43 3.62 6.43 Average 10.1 21.0 10.1 21.8 10.1 21.8 Stdev 18.1 27.5 18.1 27.8 18.1 27.8 p (t-test) 0.014 0.0091 0.0091 Min 0.0435 0.592 0.0435 0.592 0.0435 0.592 Max 111 91.6 111 91.6 111 91.6 n (Patient) 122 25 123 24 123 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.68 0.60 0.72 0.69 0.61 0.68 0.65 0.61 SE 0.045 0.046 0.065 0.046 0.047 0.066 0.050 0.050 0.066 p Value 4.9E−6 8.5E−5 0.13 3.3E−6 4.3E−5 0.10 3.1E−4 0.0031 0.10 nCohort Non-persistent 90 90 122 97 96 123 104 102 123 nCohort Persistent 58 58 25 51 52 24 44 46 24 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 84% 83% 76% 86% 85% 75% 84% 83% 75% Specificity 31% 30% 25% 31% 30% 25% 29% 28% 25% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 67% 66% 52% 69% 67% 54% 66% 63% 54% Specificity 61% 60% 50% 60% 59% 50% 57% 56% 50% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 43% 41% 40% 43% 40% 42% 43% 39% 42% Specificity 87% 86% 78% 85% 83% 78% 83% 81% 78% OR Quartile 2 2.46 2.06 1.08 2.81 2.38 1.01 2.14 1.89 1.01 p Value 0.036 0.084 0.88 0.025 0.051 0.98 0.10 0.16 0.98 Lower limit of 95% CI 1.06 0.909 0.395 1.14 0.997 0.368 0.860 0.786 0.368 Upper limit of 95% CI 5.69 4.66 2.94 6.97 5.68 2.77 5.34 4.53 2.77 OR Quartile 3 3.23 2.85 1.08 3.25 3.01 1.20 2.53 2.16 1.20 p Value 9.3E−4 0.0028 0.86 0.0013 0.0023 0.68 0.013 0.035 0.68 Lower limit of 95% CI 1.61 1.43 0.458 1.59 1.48 0.500 1.22 1.06 0.500 Upper limit of 95% CI 6.45 5.66 2.56 6.67 6.11 2.89 5.28 4.42 2.89 OR Quartile 4 4.92 4.18 2.35 4.15 3.39 2.54 3.63 2.81 2.54 p Value 9.3E−5 3.6E−4 0.065 3.6E−4 0.0019 0.046 0.0013 0.0089 0.046 Lower limit of 95% CI 2.21 1.90 0.947 1.90 1.57 1.02 1.66 1.30 1.02 Upper limit of 95% CI 11.0 9.18 5.81 9.06 7.33 6.35 7.95 6.09 6.35

TABLE 18.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.23 8.44 2.20 8.46 2.42 8.14 Average 5.84 20.1 5.84 21.1 7.59 20.2 Stdev 10.0 26.8 9.98 27.4 14.3 26.6 p (t-test) 1.3E−5 3.7E−6 2.6E−4 Min 0.0435 0.240 0.0435 0.267 0.0435 0.267 Max 57.5 111 57.5 111 103 111 n (Patient) 85 63 89 59 97 51 sCr only Median 2.30 8.29 2.23 8.44 2.57 7.37 Average 5.91 19.8 6.04 20.8 7.84 19.4 Stdev 10.1 26.7 10.1 27.5 14.4 26.6 p (t-test) 2.2E−5 8.4E−6 7.4E−4 Min 0.0435 0.172 0.0435 0.172 0.0435 0.172 Max 57.5 111 57.5 111 103 111 n (Patient) 84 64 89 59 96 52 UO only Median 3.60 8.30 3.46 8.46 3.46 8.46 Average 10.2 18.5 10.1 19.0 10.1 19.0 Stdev 18.6 24.8 18.5 25.1 18.5 25.1 p (t-test) 0.041 0.030 0.030 Min 0.0435 0.267 0.0435 0.267 0.0435 0.267 Max 111 91.6 111 91.6 111 91.6 n (Patient) 115 32 116 31 116 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.69 0.61 0.73 0.71 0.61 0.68 0.66 0.61 SE 0.044 0.045 0.058 0.044 0.045 0.059 0.048 0.048 0.059 p Value 4.0E−6 2.1E−5 0.066 1.6E−7 3.9E−6 0.054 1.1E−4 0.0013 0.054 nCohort Non-persistent 85 84 115 89 89 116 97 96 116 nCohort Persistent 63 64 32 59 59 31 51 52 31 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 84% 83% 78% 86% 85% 77% 84% 83% 77% Specificity 32% 31% 26% 33% 31% 26% 30% 29% 26% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 67% 66% 56% 69% 68% 58% 67% 63% 58% Specificity 62% 62% 51% 63% 62% 52% 59% 57% 52% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 41% 41% 38% 42% 41% 39% 41% 38% 39% Specificity 87% 87% 78% 87% 85% 78% 84% 82% 78% OR Quartile 2 2.47 2.16 1.26 3.08 2.55 1.20 2.29 1.97 1.20 p Value 0.030 0.058 0.63 0.011 0.029 0.71 0.062 0.12 0.71 Lower limit of 95% CI 1.09 0.973 0.494 1.29 1.10 0.468 0.959 0.847 0.468 Upper limit of 95% CI 5.58 4.79 3.21 7.33 5.90 3.06 5.48 4.57 3.06 OR Quartile 3 3.31 3.10 1.35 3.87 3.41 1.48 2.85 2.33 1.48 p Value 5.9E−4 0.0011 0.45 1.6E−4 5.3E−4 0.33 0.0038 0.017 0.33 Lower limit of 95% CI 1.67 1.57 0.616 1.92 1.70 0.666 1.40 1.16 0.666 Upper limit of 95% CI 6.56 6.11 2.98 7.80 6.81 3.30 5.79 4.67 3.30 OR Quartile 4 4.73 4.54 2.16 4.72 4.01 2.30 3.54 2.90 2.30 p Value 1.6E−4 2.4E−4 0.073 1.4E−4 5.2E−4 0.054 0.0013 0.0064 0.054 Lower limit of 95% CI 2.11 2.03 0.931 2.12 1.83 0.985 1.63 1.35 0.985 Upper limit of 95% CI 10.6 10.2 5.01 10.5 8.79 5.37 7.68 6.25 5.37

TABLE 18.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.22 8.29 2.20 8.46 2.42 8.14 Average 5.83 19.9 5.84 21.1 6.65 21.4 Stdev 10.1 26.7 9.98 27.4 10.6 28.6 p (t-test) 1.7E−5 3.7E−6 1.4E−5 Min 0.0435 0.240 0.0435 0.267 0.0435 0.267 Max 57.5 111 57.5 111 57.5 111 n (Patient) 84 64 89 59 95 53 sCr only Median 2.30 8.29 2.22 8.45 2.46 7.97 Average 5.91 19.8 5.90 20.7 6.72 21.0 Stdev 10.1 26.7 10.0 27.3 10.6 28.5 p (t-test) 2.2E−5 6.7E−6 2.4E−5 Min 0.0435 0.172 0.0435 0.172 0.0435 0.172 Max 57.5 111 57.5 111 57.5 111 n (Patient) 84 64 88 60 94 54 UO only Median 3.29 7.34 3.29 8.30 3.29 8.30 Average 9.60 19.3 9.51 20.2 9.51 20.2 Stdev 17.4 26.4 17.3 26.9 17.3 26.9 p (t-test) 0.012 0.0067 0.0067 Min 0.0435 0.267 0.0435 0.267 0.0435 0.267 Max 111 91.6 111 91.6 111 91.6 n (Patient) 111 36 113 34 113 34 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.63 0.73 0.71 0.63 0.69 0.68 0.63 SE 0.044 0.045 0.056 0.044 0.044 0.057 0.047 0.047 0.057 p Value 2.6E−6 2.1E−5 0.024 1.6E−7 1.2E−6 0.022 4.3E−5 1.9E−4 0.022 nCohort Non-persistent 84 84 111 89 88 113 95 94 113 nCohort Persistent 64 64 36 59 60 34 53 54 34 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 84% 83% 81% 86% 85% 79% 85% 83% 79% Specificity 32% 31% 27% 33% 32% 27% 31% 30% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 67% 66% 61% 69% 68% 62% 66% 65% 62% Specificity 63% 62% 53% 63% 62% 53% 59% 59% 53% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 41% 41% 36% 42% 42% 38% 42% 41% 38% Specificity 87% 87% 78% 87% 86% 79% 84% 84% 79% OR Quartile 2 2.56 2.16 1.53 3.08 2.64 1.39 2.47 2.12 1.39 p Value 0.024 0.058 0.36 0.011 0.023 0.48 0.041 0.080 0.48 Lower limit of 95% CI 1.13 0.973 0.608 1.29 1.14 0.550 1.04 0.915 0.550 Upper limit of 95% CI 5.78 4.79 3.87 7.33 6.12 3.53 5.90 4.92 3.53 OR Quartile 3 3.50 3.10 1.78 3.87 3.60 1.83 2.79 2.60 1.83 p Value 3.3E−4 0.0011 0.14 1.6E−4 3.0E−4 0.13 0.0041 0.0069 0.13 Lower limit of 95% CI 1.77 1.57 0.828 1.92 1.80 0.835 1.39 1.30 0.835 Upper limit of 95% CI 6.94 6.11 3.84 7.80 7.20 4.01 5.62 5.20 4.01 OR Quartile 4 4.54 4.54 2.05 4.72 4.52 2.30 3.78 3.62 2.30 p Value 2.4E−4 2.4E−4 0.085 1.4E−4 2.0E−4 0.049 7.8E−4 0.0011 0.049 Lower limit of 95% CI 2.03 2.03 0.906 2.12 2.04 1.01 1.74 1.67 1.01 Upper limit of 95% CI 10.2 10.2 4.64 10.5 10.0 5.24 8.23 7.85 5.24

TABLE 18.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.20 8.44 2.13 8.46 2.40 7.97 Average 5.32 19.9 5.34 20.8 6.69 20.5 Stdev 9.01 26.4 9.01 26.9 10.8 28.0 p (t-test) 7.4E−6 2.2E−6 3.8E−5 Min 0.0435 0.172 0.0435 0.172 0.0435 0.172 Max 57.5 111 57.5 111 57.5 111 n (Patient) 81 67 85 63 92 56 sCr only Median 2.22 8.45 2.17 8.53 2.40 8.30 Average 5.34 20.1 5.36 21.0 6.23 21.3 Stdev 8.96 26.6 8.95 27.1 9.78 28.2 p (t-test) 5.7E−6 1.6E−6 6.5E−6 Min 0.0435 0.172 0.0435 0.172 0.0435 0.172 Max 57.5 111 57.5 111 57.5 111 n (Patient) 82 66 86 62 92 56 UO only Median 3.17 8.14 3.05 8.30 3.05 8.30 Average 8.68 21.8 8.62 22.4 8.62 22.4 Stdev 14.5 30.1 14.5 30.3 14.5 30.3 p (t-test) 5.5E−4 3.4E−4 3.4E−4 Min 0.0435 0.267 0.0435 0.267 0.0435 0.267 Max 103 111 103 111 103 111 n (Patient) 110 37 111 36 111 36 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.71 0.64 0.73 0.73 0.65 0.68 0.69 0.65 SE 0.043 0.043 0.055 0.043 0.043 0.055 0.046 0.046 0.055 p Value 1.3E−6 2.1E−6 0.0097 4.6E−8 8.0E−8 0.0075 7.7E−5 2.9E−5 0.0075 nCohort Non-persistent 81 82 110 85 86 111 92 92 111 nCohort Persistent 67 66 37 63 62 36 56 56 36 Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 84% 83% 81% 86% 85% 81% 84% 84% 81% Specificity 32% 32% 27% 33% 33% 27% 30% 30% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 67% 67% 62% 70% 69% 64% 66% 66% 64% Specificity 64% 63% 54% 65% 64% 54% 60% 60% 54% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 40% 41% 38% 41% 42% 39% 39% 41% 39% Specificity 88% 88% 79% 87% 87% 79% 84% 85% 79% OR Quartile 2 2.41 2.32 1.61 2.95 2.84 1.53 2.28 2.28 1.53 p Value 0.031 0.038 0.31 0.011 0.015 0.36 0.054 0.054 0.36 Lower limit of 95% CI 1.08 1.05 0.638 1.27 1.23 0.608 0.986 0.986 0.608 Upper limit of 95% CI 5.34 5.15 4.05 6.81 6.57 3.87 5.29 5.29 3.87 OR Quartile 3 3.67 3.47 1.90 4.25 4.02 2.08 2.89 2.89 2.08 p Value 1.9E−4 3.5E−4 0.099 4.9E−5 9.2E−5 0.064 0.0026 0.0026 0.064 Lower limit of 95% CI 1.85 1.75 0.886 2.11 2.00 0.958 1.45 1.45 0.958 Upper limit of 95% CI 7.26 6.85 4.08 8.53 8.06 4.52 5.79 5.79 4.52 OR Quartile 4 4.79 4.98 2.30 4.73 4.92 2.43 3.32 3.88 2.43 p Value 1.9E−4 1.3E−4 0.043 1.6E−4 1.1E−4 0.032 0.0023 6.4E−4 0.032 Lower limit of 95% CI 2.11 2.19 1.03 2.11 2.19 1.08 1.54 1.78 1.08 Upper limit of 95% CI 10.9 11.4 5.16 10.6 11.1 5.49 7.18 8.46 5.49

TABLE 18.6 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 90.7 129 94.2 135 94.2 136 Average 102 133 103 136 105 137 Stdev 53.6 79.7 55.0 81.2 55.4 85.0 p (t-test) 0.0056 0.0045 0.0085 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 95 55 103 47 110 40 sCr only Median 92.4 128 94.2 129 95.2 124 Average 103 131 104 133 107 131 Stdev 53.5 80.0 55.4 81.5 56.1 84.5 p (t-test) 0.010 0.012 0.044 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 94 56 103 47 108 42 UO only Median 97.5 157 97.5 157 98.9 148 Average 108 155 108 155 108 154 Stdev 59.5 91.2 59.5 91.2 59.5 93.8 p (t-test) 0.0034 0.0034 0.0056 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 284 401 284 401 284 401 n (Patient) 130 19 130 19 131 18 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.61 0.60 0.66 0.62 0.60 0.66 0.60 0.57 0.65 SE 0.049 0.049 0.072 0.051 0.051 0.072 0.054 0.053 0.074 p Value 0.021 0.041 0.022 0.021 0.054 0.022 0.057 0.20 0.041 nCohort Non-persistent 95 94 130 103 103 130 110 108 131 nCohort Persistent 55 56 19 47 47 19 40 42 18 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 80% 79% 84% 81% 79% 84% 80% 76% 83% Specificity 28% 28% 26% 28% 27% 26% 27% 26% 26% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 62% 61% 68% 62% 62% 68% 62% 60% 67% Specificity 57% 56% 52% 55% 55% 52% 55% 54% 52% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 36% 36% 53% 36% 34% 53% 38% 33% 50% Specificity 81% 81% 78% 80% 79% 78% 79% 78% 78% OR Quartile 2 1.59 1.40 1.89 1.65 1.38 1.89 1.50 1.12 1.75 p Value 0.26 0.40 0.34 0.24 0.44 0.34 0.37 0.79 0.40 Lower limit of 95% CI 0.716 0.641 0.518 0.711 0.607 0.518 0.621 0.488 0.478 Upper limit of 95% CI 3.52 3.06 6.89 3.85 3.14 6.89 3.62 2.57 6.43 OR Quartile 3 2.13 2.00 2.38 2.00 2.00 2.38 2.00 1.71 2.16 p Value 0.029 0.044 0.098 0.055 0.055 0.098 0.067 0.15 0.15 Lower limit of 95% CI 1.08 1.02 0.851 0.987 0.987 0.851 0.952 0.828 0.764 Upper limit of 95% CI 4.20 3.92 6.63 4.04 4.04 6.63 4.20 3.52 6.10 OR Quartile 4 2.44 2.35 4.05 2.21 1.90 4.05 2.27 1.75 3.52 p Value 0.020 0.026 0.0058 0.042 0.10 0.0058 0.041 0.16 0.015 Lower limit of 95% CI 1.15 1.11 1.50 1.03 0.884 1.50 1.03 0.798 1.28 Upper limit of 95% CI 5.18 4.97 10.9 4.75 4.09 10.9 4.99 3.84 9.67

TABLE 18.7 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 89.6 121 92.5 120 94.3 118 Average 101 131 103 131 105 131 Stdev 53.2 77.7 54.7 78.7 55.1 81.7 p (t-test) 0.0063 0.012 0.025 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 88 62 94 56 101 49 sCr only Median 92.5 119 96.1 113 98.9 111 Average 103 127 105 126 108 124 Stdev 55.2 76.9 56.7 77.4 56.9 79.9 p (t-test) 0.029 0.059 0.15 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 253 401 253 401 253 401 n (Patient) 88 62 93 57 99 51 UO only Median 101 124 101 124 101 111 Average 109 137 109 137 109 136 Stdev 58.7 90.7 58.7 90.7 58.7 92.3 p (t-test) 0.046 0.046 0.067 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 284 401 284 401 284 401 n (Patient) 123 26 123 26 124 25 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.61 0.59 0.58 0.60 0.57 0.58 0.58 0.54 0.56 SE 0.047 0.048 0.064 0.049 0.049 0.064 0.051 0.050 0.065 p Value 0.021 0.072 0.22 0.046 0.15 0.22 0.12 0.41 0.32 nCohort Non-persistent 88 88 123 94 93 123 101 99 124 nCohort Persistent 62 62 26 56 57 26 49 51 25 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 82% 81% 81% 82% 81% 81% 82% 78% 80% Specificity 31% 30% 26% 30% 29% 26% 29% 27% 26% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 60% 58% 54% 59% 56% 54% 59% 55% 52% Specificity 57% 56% 50% 55% 54% 50% 54% 53% 50% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 34% 32% 42% 32% 30% 42% 33% 29% 40% Specificity 81% 80% 78% 79% 77% 78% 78% 77% 77% OR Quartile 2 2.05 1.75 1.48 1.95 1.71 1.48 1.79 1.36 1.39 p Value 0.076 0.16 0.47 0.11 0.19 0.47 0.18 0.45 0.54 Lower limit of 95% CI 0.928 0.802 0.514 0.864 0.772 0.514 0.771 0.612 0.482 Upper limit of 95% CI 4.54 3.81 4.24 4.41 3.79 4.24 4.15 3.04 4.01 OR Quartile 3 1.95 1.74 1.19 1.78 1.49 1.19 1.73 1.35 1.08 p Value 0.048 0.098 0.69 0.093 0.24 0.69 0.12 0.39 0.86 Lower limit of 95% CI 1.01 0.902 0.508 0.909 0.767 0.508 0.868 0.684 0.458 Upper limit of 95% CI 3.77 3.36 2.77 3.47 2.89 2.77 3.46 2.65 2.56 OR Quartile 4 2.14 1.85 2.61 1.75 1.46 2.61 1.74 1.38 2.29 p Value 0.046 0.10 0.034 0.14 0.32 0.034 0.15 0.41 0.073 Lower limit of 95% CI 1.01 0.881 1.07 0.830 0.690 1.07 0.813 0.643 0.925 Upper limit of 95% CI 4.51 3.89 6.33 3.70 3.08 6.33 3.73 2.95 5.65

TABLE 18.8 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 88.6 122 89.6 124 94.2 120 Average 98.0 132 98.0 134 102 132 Stdev 50.6 77.1 50.5 77.9 53.8 79.5 p (t-test) 0.0013 8.3E−4 0.0083 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 253 401 n (Patient) 83 67 86 64 94 56 sCr only Median 89.6 121 92.5 120 96.1 113 Average 98.7 131 99.8 131 105 127 Stdev 50.5 77.3 51.3 78.3 54.4 79.6 p (t-test) 0.0026 0.0035 0.038 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 253 401 n (Patient) 82 68 86 64 93 57 UO only Median 99.1 121 99.1 121 99.8 113 Average 107 135 107 135 108 134 Stdev 58.0 85.1 58.0 85.1 58.0 86.2 p (t-test) 0.028 0.028 0.041 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 284 401 284 401 284 401 n (Patient) 115 34 115 34 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.63 0.62 0.59 0.63 0.61 0.59 0.60 0.57 0.58 SE 0.046 0.046 0.057 0.046 0.047 0.057 0.048 0.049 0.058 p Value 0.0046 0.0099 0.13 0.0039 0.017 0.13 0.039 0.16 0.19 nCohort 83 82 115 86 86 115 94 93 116 Non-persistent nCohort 67 68 34 64 64 34 56 57 33 Persistent Cutoff 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Quartile 2 Sensitivity 84% 82% 82% 84% 83% 82% 84% 81% 82% Specificity 33% 32% 27% 33% 31% 27% 31% 29% 27% Cutoff 101 101 101 101 101 101 101 101 101 Quartile 3 Sensitivity 60% 59% 56% 59% 58% 56% 59% 56% 55% Specificity 58% 57% 51% 57% 56% 51% 55% 54% 51% Cutoff 142 142 142 142 142 142 142 142 142 Quartile 4 Sensitivity 34% 34% 38% 34% 33% 38% 32% 30% 36% Specificity 82% 82% 78% 81% 80% 78% 79% 77% 78% OR Quartile 2 2.45 2.17 1.72 2.61 2.20 1.72 2.33 1.71 1.64 p Value 0.026 0.051 0.27 0.021 0.051 0.27 0.048 0.19 0.32 Lower limit 1.11 0.995 0.651 1.16 0.998 0.651 1.01 0.772 0.619 of 95% CI Upper limit 5.42 4.72 4.56 5.87 4.87 4.56 5.38 3.79 4.35 of 95% CI OR Quartile 3 2.03 1.92 1.33 1.94 1.73 1.33 1.78 1.49 1.24 p Value 0.034 0.050 0.46 0.049 0.100 0.46 0.093 0.24 0.58 Lower limit 1.06 1.000 0.618 1.00 0.900 0.618 0.909 0.767 0.572 of 95% CI Upper limit 3.91 3.68 2.88 3.73 3.33 2.88 3.47 2.89 2.70 of 95% CI OR Quartile 4 2.37 2.28 2.23 2.29 1.98 2.23 1.75 1.46 1.98 p Value 0.025 0.031 0.056 0.030 0.072 0.056 0.14 0.32 0.11 Lower limit 1.12 1.08 0.980 1.08 0.942 0.980 0.830 0.690 0.860 of 95% CI Upper limit 5.03 4.84 5.07 4.85 4.17 5.07 3.70 3.08 4.55 of 95% CI

TABLE 18.9 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non- Non- Non- persistent Persistent persistent Persistent persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 87.0 122 88.6 127 94.2 122 Average 96.4 133 96.5 135 100 133 Stdev 49.1 77.0 48.8 78.2 51.3 79.8 p (t-test) 5.4E−4 2.7E−4 0.0023 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 253 401 n (Patient) 81 69 85 65 91 59 sCr only Median 88.6 122 89.6 124 94.2 120 Average 97.1 132 97.1 134 101 132 Stdev 48.7 77.7 48.7 78.4 51.2 80.1 p (t-test) 9.4E−4 5.8E−4 0.0043 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 253 401 n (Patient) 81 69 84 66 90 60 UO only Median 94.3 123 95.2 129 96.1 123 Average 104 139 104 140 105 139 Stdev 55.7 83.5 55.5 84.3 55.6 85.3 p (t-test) 0.0038 0.0030 0.0048 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 274 401 274 401 274 401 n (Patient) 109 40 110 39 111 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.63 0.62 0.64 0.63 0.62 0.61 0.60 0.61 SE 0.045 0.046 0.054 0.046 0.046 0.054 0.048 0.048 0.055 p Value 0.0019 0.0044 0.027 0.0016 0.0037 0.026 0.016 0.031 0.043 nCohort 81 81 109 85 84 110 91 90 111 Non-persistent nCohort 69 69 40 65 66 39 59 60 38 Persistent Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 84% 83% 85% 85% 83% 85% 83% 82% 84% Specificity 33% 32% 28% 33% 32% 28% 31% 30% 28% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 59% 59% 60% 60% 59% 62% 59% 58% 61% Specificity 58% 58% 53% 58% 57% 54% 56% 56% 53% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 35% 35% 38% 35% 35% 38% 34% 33% 37% Specificity 83% 83% 79% 82% 82% 79% 80% 80% 78% OR Quartile 2 2.64 2.25 2.25 2.70 2.37 2.16 2.18 1.91 2.07 p Value 0.017 0.042 0.098 0.016 0.033 0.12 0.061 0.11 0.14 Lower limit 1.19 1.03 0.860 1.20 1.07 0.823 0.966 0.863 0.787 of 95% CI Upper limit 5.83 4.89 5.90 6.08 5.23 5.66 4.91 4.22 5.43 of 95% CI OR Quartile 3 2.02 2.02 1.71 2.04 1.93 1.85 1.86 1.75 1.74 p Value 0.034 0.034 0.15 0.033 0.049 0.11 0.067 0.097 0.15 Lower limit 1.05 1.05 0.817 1.06 1.00 0.878 0.957 0.904 0.822 of 95% CI Upper limit 3.89 3.89 3.56 3.94 3.70 3.90 3.61 3.39 3.68 of 95% CI OR Quartile 4 2.55 2.55 2.24 2.56 2.46 2.36 2.08 2.00 2.11 p Value 0.016 0.016 0.045 0.015 0.019 0.033 0.054 0.068 0.066 Lower limit 1.19 1.19 1.02 1.20 1.16 1.07 0.986 0.950 0.951 of 95% CI Upper limit 5.46 5.46 4.94 5.44 5.23 5.22 4.39 4.21 4.70 of 95% CI

TABLE 18.10 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 86.1 121 87.0 122 92.8 120 Average 96.0 132 96.0 133 100.0 132 Stdev 48.8 76.4 48.7 77.1 51.1 79.1 p (t-test) 6.8E−4 4.2E−4 0.0029 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 253 401 n (Patient) 78 72 81 69 88 62 sCr only Median 87.0 122 87.8 124 92.8 120 Average 96.0 132 96.1 134 100.0 132 Stdev 48.4 76.9 48.4 77.6 51.1 79.1 p (t-test) 5.9E−4 3.6E−4 0.0029 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 246 401 246 401 253 401 n (Patient) 79 71 82 68 88 62 UO only Median 94.2 129 94.2 129 94.3 123 Average 103 143 103 143 103 142 Stdev 53.5 85.1 53.5 85.1 53.7 86.0 p (t-test) 8.4E−4 8.4E−4 0.0014 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 260 401 260 401 260 401 n (Patient) 108 41 108 41 109 40 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.64 0.63 0.64 0.64 0.63 0.61 0.61 0.62 SE 0.045 0.045 0.053 0.045 0.046 0.053 0.047 0.047 0.053 p Value 0.0022 0.0022 0.012 0.0019 0.0018 0.012 0.019 0.019 0.021 nCohort 78 79 108 81 82 108 88 88 109 Non-persistent nCohort 72 71 41 69 68 41 62 62 40 Persistent Cutoff 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Quartile 2 Sensitivity 83% 83% 85% 84% 84% 85% 82% 82% 85% Specificity 33% 33% 29% 33% 33% 29% 31% 31% 28% Cutoff 101 101 101 101 101 101 101 101 101 Quartile 3 Sensitivity 58% 59% 61% 58% 59% 61% 58% 58% 60% Specificity 58% 58% 54% 57% 57% 54% 56% 56% 53% Cutoff 142 142 142 142 142 142 142 142 142 Quartile 4 Sensitivity 35% 35% 39% 35% 35% 39% 34% 34% 38% Specificity 83% 84% 80% 83% 83% 80% 81% 81% 79% OR Quartile 2 2.50 2.41 2.35 2.64 2.54 2.35 2.05 2.05 2.25 p Value 0.021 0.027 0.082 0.017 0.021 0.082 0.076 0.076 0.098 Lower limit 1.15 1.11 0.898 1.19 1.15 0.898 0.928 0.928 0.860 of 95% CI Upper limit 5.44 5.25 6.14 5.83 5.62 6.14 4.54 4.54 5.90 of 95% CI OR Quartile 3 1.91 2.02 1.81 1.81 1.92 1.81 1.74 1.74 1.71 p Value 0.051 0.034 0.11 0.073 0.050 0.11 0.098 0.098 0.15 Lower limit 0.998 1.05 0.871 0.947 1.000 0.871 0.902 0.902 0.817 of 95% CI Upper limit 3.65 3.87 3.77 3.47 3.68 3.77 3.36 3.36 3.56 of 95% CI OR Quartile 4 2.66 2.76 2.50 2.55 2.65 2.50 2.14 2.14 2.24 p Value 0.013 0.0097 0.022 0.016 0.012 0.022 0.046 0.046 0.045 Lower limit 1.23 1.28 1.14 1.19 1.24 1.14 1.01 1.01 1.02 of 95% CI Upper limit 5.73 5.95 5.47 5.46 5.67 5.47 4.51 4.51 4.94 of 95% CI

Example 19. Use of Cathepsin B for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Cathepsin B is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 19.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.57 7.50 2.42 6.67 2.42 8.44 Average 2.92 17.1 6.68 17.6 6.52 19.6 Stdev 3.60 23.8 10.8 25.9 10.4 27.4 p (t-test) 2.7E−5 9.0E−4 7.7E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 54 94 77 71 87 61 sCr only Median 1.74 7.97 2.37 7.81 2.29 8.53 Average 3.01 17.3 5.59 18.4 5.56 21.2 Stdev 3.56 24.0 9.35 25.8 8.93 27.5 p (t-test) 1.9E−5 8.4E−5 1.8E−6 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 56 92 75 73 88 60 UO only Median 3.29 5.69 3.46 8.62 3.46 8.62 Average 10.6 16.7 10.2 19.8 10.1 22.0 Stdev 19.0 24.0 18.5 26.1 18.2 27.6 p (t-test) 0.12 0.026 0.0095 Min 0.0435 0.592 0.0435 0.592 0.0435 0.592 Max 111 91.6 111 91.6 111 91.6 n (Patient) 113 34 120 27 124 23 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.74 0.61 0.66 0.68 0.64 0.68 0.71 0.65 SE 0.039 0.040 0.057 0.045 0.044 0.062 0.045 0.044 0.066 p Value 1.2E−10 1.5E−9 0.049 5.1E−4 4.8E−5 0.029 7.8E−5 2.2E−6 0.028 nCohort 54 56 113 77 75 120 87 88 124 Non-persistent nCohort 94 92 34 71 73 27 61 60 23 Persistent Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 83% 83% 85% 83% 84% 85% 85% 87% 83% Specificity 39% 38% 28% 32% 33% 28% 32% 33% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 64% 63% 62% 62% 63% 59% 64% 67% 61% Specificity 74% 71% 53% 61% 63% 52% 60% 61% 52% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 36% 37% 32% 34% 37% 41% 38% 42% 43% Specificity 94% 95% 77% 83% 87% 78% 84% 86% 78% OR Quartile 2 3.10 2.85 2.29 2.36 2.54 2.18 2.74 3.19 1.72 p Value 0.0038 0.0072 0.12 0.031 0.020 0.18 0.018 0.0086 0.35 Lower limit 1.44 1.33 0.815 1.08 1.16 0.701 1.19 1.34 0.546 of 95% CI Upper limit 6.68 6.12 6.44 5.17 5.56 6.78 6.34 7.60 5.44 of 95% CI OR Quartile 3 5.04 4.26 1.83 2.55 2.86 1.55 2.63 3.18 1.66 p Value 1.8E−5  7.5E−5 0.13 0.0056 0.0020 0.31 0.0050 9.8E−4 0.27 Lower limit 2.41 2.08 0.835 1.32 1.47 0.667 1.34 1.60 0.669 of 95% CI Upper limit 10.6 8.74 4.01 4.95 5.57 3.63 5.18 6.32 4.12 of 95% CI OR Quartile 4 9.63 10.4 1.60 2.51 3.82 2.49 3.16 4.52 2.76 p Value 3.4E−4  2.1E−4 0.27 0.019 0.0013 0.043 0.0035 2.0E−4 0.032 Lower limit 2.79 3.00 0.690 1.16 1.68 1.03 1.46 2.04 1.09 of 95% CI Upper limit 33.2 35.7 3.71 5.45 8.64 6.00 6.83 10.0 6.99 of 95% CI

TABLE 19.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.55 7.19 2.17 7.37 2.13 8.46 Average 2.90 16.9 5.39 18.1 5.35 19.9 Stdev 3.63 23.8 9.40 25.3 9.10 26.4 p (t-test) 3.5E−5 1.0E−4 8.2E−6 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 53 95 72 76 81 67 sCr only Median 1.60 7.81 2.29 7.37 2.20 8.44 Average 2.99 17.2 5.48 18.0 5.50 19.7 Stdev 3.59 23.9 9.38 25.4 9.13 26.5 p (t-test) 2.4E−5 1.3E−4 1.4E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 55 93 72 76 81 67 UO only Median 3.05 6.73 3.30 8.62 3.30 8.62 Average 9.45 18.3 9.15 21.1 9.08 22.8 Stdev 17.6 25.1 17.1 26.6 16.8 27.7 p (t-test) 0.016 0.0022 6.8E−4 Min 0.0435 0.592 0.0435 0.592 0.0435 0.592 Max 111 91.6 111 91.6 111 91.6 n (Patient) 105 42 112 35 116 31 Persistence Period Duration (hr) 24 48 72 UO sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only only AUC 0.76 0.74 0.63 0.70 0.68 0.65 0.72 0.70 0.66 SE 0.039 0.040 0.053 0.043 0.044 0.056 0.043 0.044 0.058 p Value 7.0E−11 9.2E−10 0.011 4.1E−6 3.3E−5 0.0065 1.9E−7 3.2E−6 0.0064 nCohort 53 55 105 72 72 112 81 81 116 Non-persistent nCohort 95 93 42 76 76 35 67 67 31 Persistent Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 83% 83% 83% 84% 83% 83% 87% 85% 81% Specificity 40% 38% 29% 35% 33% 28% 35% 33% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 64% 63% 62% 64% 63% 60% 67% 66% 61% Specificity 75% 73% 54% 65% 64% 53% 64% 63% 53% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 36% 37% 36% 37% 37% 43% 40% 39% 45% Specificity 94% 95% 79% 88% 88% 80% 88% 86% 80% OR Quartile 2 3.24 2.97 2.00 2.84 2.42 1.85 3.40 2.85 1.52 p Value 0.0027 0.0053 0.14 0.0092 0.025 0.21 0.0042 0.012 0.40 Lower limit 1.50 1.38 0.801 1.29 1.12 0.700 1.47 1.26 0.570 of 95% CI Upper limit 6.99 6.39 5.00 6.22 5.25 4.89 7.87 6.44 4.05 of 95% CI OR Quartile 3 5.52 4.63 1.93 3.41 3.03 1.67 3.67 3.25 1.76 p Value 8.8E−6  3.7E−5  0.078 3.7E−4 0.0012 0.19 1.9E−4 6.3E−4 0.17 Lower limit 2.60 2.23 0.928 1.74 1.55 0.772 1.85 1.65 0.782 of 95% CI Upper limit 11.7 9.58 4.01 6.70 5.93 3.61 7.26 6.40 3.95 of 95% CI OR Quartile 4 9.29 9.99 2.10 4.08 4.08 3.07 4.79 4.04 3.33 p Value 4.2E−4  2.7E−4  0.065 0.0010 0.0010 0.0071 1.9E−4 6.7E−4 0.0051 Lower limit 2.69 2.90 0.954 1.76 1.76 1.36 2.11 1.81 1.43 of 95% CI Upper limit 32.0 34.4 4.60 9.46 9.46 6.94 10.9 9.01 7.73 of 95% CI

TABLE 19.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.55 7.19 2.13 6.94 2.13 8.14 Average 2.90 16.9 5.41 17.9 5.45 18.9 Stdev 3.63 23.8 9.46 25.2 9.30 25.9 p (t-test) 3.5E−5 1.3E−4 3.5E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 53 95 71 77 77 71 sCr only Median 1.60 7.81 2.20 6.94 2.20 8.14 Average 2.99 17.2 5.50 17.8 5.53 18.8 Stdev 3.59 23.9 9.44 25.3 9.28 26.0 p (t-test) 2.4E−5 1.7E−4 4.4E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 55 93 71 77 77 71 UO only Median 3.05 6.73 3.29 8.30 3.29 8.30 Average 9.69 16.7 9.36 18.6 9.27 19.8 Stdev 18.1 23.9 17.6 25.0 17.3 25.9 p (t-test) 0.049 0.013 0.0058 Min 0.0435 0.267 0.0435 0.267 0.0435 0.267 Max 111 91.6 111 91.6 111 91.6 n (Patient) 99 48 105 42 109 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.76 0.74 0.61 0.70 0.68 0.63 0.71 0.69 0.63 SE 0.039 0.040 0.051 0.043 0.044 0.053 0.043 0.043 0.054 p Value 7.0E−11 9.2E−10 0.026 3.5E−6 2.9E−5 0.013 9.9E−7 9.3E−6 0.014 nCohort 53 55 99 71 71 105 77 77 109 Non-persistent nCohort 95 93 48 77 77 42 71 71 38 Persistent Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 83% 83% 81% 84% 83% 81% 86% 85% 79% Specificity 40% 38% 28% 35% 34% 28% 35% 34% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 64% 63% 60% 65% 64% 60% 66% 65% 61% Specificity 75% 73% 55% 66% 65% 53% 65% 64% 53% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 36% 37% 33% 36% 36% 38% 38% 38% 39% Specificity 94% 95% 79% 87% 87% 80% 87% 87% 80% OR Quartile 2 3.24 2.97 1.71 2.94 2.51 1.62 3.29 2.78 1.36 p Value 0.0027 0.0053 0.21 0.0070 0.019 0.28 0.0042 0.012 0.50 Lower limit 1.50 1.38 0.733 1.34 1.16 0.672 1.46 1.25 0.559 of 95% CI Upper limit 6.99 6.39 3.98 6.45 5.45 3.91 7.45 6.17 3.30 of 95% CI OR Quartile 3 5.52 4.63 1.83 3.63 3.22 1.68 3.63 3.22 1.74 p Value 8.8E−6  3.7E−5  0.091 2.0E−4 6.6E−4 0.16 2.0E−4 6.6E−4 0.15 Lower limit 2.60 2.23 0.909 1.84 1.64 0.813 1.84 1.64 0.823 of 95% CI Upper limit 11.7 9.58 3.69 7.15 6.31 3.47 7.15 6.31 3.70 of 95% CI OR Quartile 4 9.29 9.99 1.86 3.94 3.94 2.46 4.11 4.11 2.58 p Value 4.2E−4  2.7E−4  0.11 0.0014 0.0014 0.025 7.2E−4 7.2E−4 0.020 Lower limit 2.69 2.90 0.860 1.70 1.70 1.12 1.81 1.81 1.16 of 95% CI Upper limit of 32.0 34.4 4.01 9.11 9.11 5.40 9.33 9.33 5.75 95% CI

TABLE 19.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.55 7.19 2.13 6.94 2.13 8.14 Average 2.90 16.9 5.41 17.9 5.45 18.9 Stdev 3.63 23.8 9.46 25.2 9.30 25.9 p (t-test) 3.5E−5 1.3E−4 3.5E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 53 95 71 77 77 71 sCr only Median 1.60 7.81 2.20 6.94 2.20 8.14 Average 2.99 17.2 5.50 17.8 5.53 18.8 Stdev 3.59 23.9 9.44 25.3 9.28 26.0 p (t-test) 2.4E−5 1.7E−4 4.4E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 55 93 71 77 77 71 UO only Median 3.05 6.20 3.17 7.81 3.17 7.81 Average 9.81 16.2 9.51 17.6 9.42 18.6 Stdev 18.2 23.5 17.8 24.4 17.5 25.3 p (t-test) 0.071 0.026 0.013 Min 0.0435 0.267 0.0435 0.267 0.0435 0.267 Max 111 91.6 111 91.6 111 91.6 n (Patient) 97 50 102 45 106 41 Persistence Period Duration (hr) 24 48 72 UO sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only only AUC 0.76 0.74 0.61 0.70 0.68 0.62 0.71 0.69 0.63 SE 0.039 0.040 0.050 0.043 0.044 0.051 0.043 0.043 0.053 p Value 7.0E−11 9.2E−10 0.031 3.5E−6 2.9E−5 0.015 9.9E−7 9.3E−6 0.017 nCohort 53 55 97 71 71 102 77 77 106 Non-persistent nCohort 95 93 50 77 77 45 71 71 41 Persistent Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 83% 83% 82% 84% 83% 82% 86% 85% 80% Specificity 40% 38% 29% 35% 34% 28% 35% 34% 27% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 64% 63% 60% 65% 64% 60% 66% 65% 61% Specificity 75% 73% 55% 66% 65% 54% 65% 64% 54% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 36% 37% 32% 36% 36% 36% 38% 38% 37% Specificity 94% 95% 78% 87% 87% 79% 87% 87% 79% OR Quartile 2 3.24 2.97 1.85 2.94 2.51 1.84 3.29 2.78 1.55 p Value 0.0027 0.0053 0.15 0.0070 0.019 0.17 0.0042 0.012 0.33 Lower limit 1.50 1.38 0.794 1.34 1.16 0.764 1.46 1.25 0.643 of 95% CI Upper limit 6.99 6.39 4.30 6.45 5.45 4.42 7.45 6.17 3.76 of 95% CI OR Quartile 3 5.52 4.63 1.81 3.63 3.22 1.76 3.63 3.22 1.82 p Value 8.8E−6  3.7E−5  0.094 2.0E−4 6.6E−4 0.12 2.0E−4 6.6E−4 0.11 Lower limit 2.60 2.23 0.904 1.84 1.64 0.861 1.84 1.64 0.872 of 95% CI Upper limit 11.7 9.58 3.61 7.15 6.31 3.58 7.15 6.31 3.79 of 95% CI OR Quartile 4 9.29 9.99 1.70 3.94 3.94 2.13 4.11 4.11 2.20 p Value 4.2E−4  2.7E−4  0.17 0.0014 0.0014 0.057 7.2E−4 7.2E−4 0.050 Lower limit 2.69 2.90 0.792 1.70 1.70 0.979 1.81 1.81 1.000 of 95% CI Upper limit 32.0 34.4 3.66 9.11 9.11 4.63 9.33 9.33 4.85 of 95% CI

TABLE 19.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.55 7.19 2.17 6.80 2.13 7.81 Average 2.73 16.8 5.50 17.4 5.54 18.1 Stdev 3.28 23.6 9.62 24.9 9.51 25.5 p (t-test) 4.2E−5 3.1E−4 1.2E−4 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 51 97 68 80 73 75 sCr only Median 1.57 7.97 2.13 7.81 2.13 8.44 Average 2.78 17.2 5.32 17.7 5.36 18.7 Stdev 3.25 23.8 9.50 25.0 9.34 25.7 p (t-test) 2.0E−5 1.6E−4 4.5E−5 Min 0.0435 0.156 0.0435 0.156 0.0435 0.156 Max 17.5 111 57.5 111 57.5 111 n (Patient) 54 94 69 79 75 73 UO only Median 3.03 6.73 3.04 7.81 3.03 7.97 Average 8.75 17.9 8.61 18.7 8.55 19.5 Stdev 15.1 26.6 14.9 27.2 14.7 27.8 p (t-test) 0.0087 0.0041 0.0021 Min 0.0435 0.267 0.0435 0.267 0.0435 0.267 Max 103 111 103 111 103 111 n (Patient) 95 52 98 49 101 46 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.75 0.63 0.68 0.70 0.63 0.69 0.71 0.64 SE 0.039 0.039 0.049 0.043 0.043 0.050 0.043 0.043 0.051 p Value 1.2E−10 9.8E−11 0.0095 2.6E−5 4.8E−6 0.0082 6.9E−6 1.5E−6 0.0065 nCohort 51 54 95 68 69 98 73 75 101 Non-persistent nCohort 97 94 52 80 79 49 75 73 46 Persistent Cutoff Quartile 2 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 1.13 Sensitivity 82% 83% 83% 82% 84% 82% 84% 85% 80% Specificity 39% 39% 29% 34% 35% 29% 34% 35% 28% Cutoff Quartile 3 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 3.63 Sensitivity 64% 64% 62% 64% 65% 61% 65% 66% 63% Specificity 76% 74% 56% 66% 67% 55% 66% 65% 55% Cutoff Quartile 4 12.8 12.8 13.1 12.8 12.8 13.1 12.8 12.8 13.1 Sensitivity 36% 37% 33% 35% 37% 35% 36% 38% 35% Specificity 96% 96% 79% 87% 88% 80% 86% 88% 79% OR Quartile 2 3.04 3.10 2.00 2.41 2.71 1.78 2.73 2.99 1.58 p Value 0.0046 0.0038 0.11 0.024 0.012 0.18 0.012 0.0072 0.29 Lower limit 1.41 1.44 0.859 1.12 1.25 0.763 1.25 1.35 0.675 of 95% CI Upper limit 6.54 6.68 4.64 5.18 5.87 4.14 5.99 6.64 3.68 of 95% CI OR Quartile 3 5.76 5.04 2.02 3.44 3.64 1.94 3.62 3.62 2.12 p Value 8.0E−6 1.8E−5 0.046 3.6E−4 2.0E−4 0.064 2.0E−4 2.0E−4 0.039 Lower limit 2.67 2.41 1.01 1.75 1.84 0.963 1.84 1.84 1.04 of 95% CI Upper limit 12.4 10.6 4.03 6.78 7.19 3.90 7.13 7.13 4.34 of 95% CI OR Quartile 4 13.8 15.4 1.82 3.53 4.42 2.07 3.54 4.56 2.03 p Value 4.8E−4 2.7E−4 0.12 0.0032 7.8E−4 0.062 0.0024 4.0E−4 0.073 Lower limit 3.17 3.54 0.851 1.53 1.86 0.963 1.57 1.97 0.937 of 95% CI Upper limit 60.4 67.3 3.90 8.16 10.5 4.46 8.02 10.6 4.41 of 95% CI

TABLE 19.6 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non- Non-persistent Persistent Non-persistent Persistent persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 67.7 118 88.6 113 91.4 118 Average 83.0 128 100 126 101 130 Stdev 42.9 70.2 54.3 73.9 51.8 78.0 p (t-test) 4.6E−5 0.017 0.0068 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 174 401 251 401 251 401 n (Patient) 50 100 75 75 85 65 sCr only Median 77.0 117 88.6 118 92.8 120 Average 87.3 127 99.0 127 101 129 Stdev 44.7 71.1 51.5 74.9 52.8 77.7 p (t-test) 3.4E−4 0.0095 0.0095 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 182 401 246 401 253 401 n (Patient) 52 98 73 77 86 64 UO only Median 90.0 136 95.2 136 96.1 132 Average 106 137 107 139 108 142 Stdev 61.7 73.4 60.7 80.5 59.9 87.1 p (t-test) 0.014 0.019 0.020 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 284 401 284 401 284 401 n (Patient) 112 37 120 29 125 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.67 0.64 0.60 0.60 0.63 0.60 0.60 0.62 SE 0.043 0.044 0.055 0.046 0.046 0.060 0.047 0.047 0.066 p Value 1.5E−6 1.2E−4 0.0098 0.029 0.024 0.035 0.030 0.039 0.075 nCohort 50 52 112 75 73 120 85 86 125 Non-persistent nCohort 100 98 37 75 77 29 65 64 24 Persistent Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 85% 84% 86% 81% 81% 86% 80% 80% 83% Specificity 46% 42% 29% 32% 32% 28% 29% 29% 26% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 59% 58% 68% 55% 56% 62% 57% 58% 62% Specificity 68% 65% 55% 55% 56% 52% 55% 56% 52% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 32% 31% 38% 31% 31% 41% 34% 33% 42% Specificity 88% 85% 79% 80% 81% 78% 81% 80% 78% OR Quartile 2 4.83 3.76 2.56 2.05 1.90 2.37 1.67 1.61 1.79 p Value 7.9E−5 7.3E−4 0.073 0.063 0.093 0.13 0.19 0.22 0.32 Lower limit 2.21 1.74 0.916 0.962 0.898 0.767 0.775 0.747 0.571 of 95% CI Upper limit 10.5 8.10 7.16 4.37 4.02 7.33 3.59 3.46 5.63 of 95% CI OR Quartile 3 3.06 2.63 2.58 1.45 1.62 1.81 1.63 1.73 1.81 p Value 0.0022 0.0067 0.017 0.25 0.14 0.16 0.14 0.100 0.20 Lower limit 1.50 1.31 1.18 0.765 0.850 0.788 0.852 0.900 0.736 of 95% CI Upper limit 6.25 5.28 5.65 2.77 3.09 4.15 3.13 3.33 4.43 of 95% CI OR Quartile 4 3.45 2.43 2.23 1.77 1.91 2.55 2.21 1.98 2.47 p Value 0.011 0.045 0.050 0.14 0.094 0.032 0.038 0.072 0.052 Lower limit 1.33 1.02 1.000 0.837 0.896 1.08 1.04 0.942 0.992 of 95% CI Upper limit 8.93 5.78 4.98 3.74 4.07 6.01 4.66 4.17 6.17 of 95% CI

TABLE 19.7 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 67.1 119 86.1 115 88.6 118 Average 81.4 129 97.7 127 98.3 130 Stdev 41.9 69.9 52.5 73.3 50.3 76.7 p (t-test) 2.3E−5 0.0062 0.0031 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 174 401 246 401 246 401 n (Patient) 49 101 70 80 79 71 sCr only Median 73.7 118 87.8 115 94.3 113 Average 85.8 127 98.5 126 101 127 Stdev 43.9 70.8 52.1 73.8 52.8 76.0 p (t-test) 1.9E−4 0.0097 0.017 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 182 401 246 401 253 401 n (Patient) 51 99 70 80 79 71 UO only Median 91.4 125 96.1 117 97.5 113 Average 105 134 106 136 107 137 Stdev 59.4 75.2 58.4 81.0 57.7 85.9 p (t-test) 0.011 0.016 0.020 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 274 401 274 401 274 401 n (Patient) 103 46 111 38 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.68 0.63 0.62 0.61 0.61 0.62 0.59 0.59 SE 0.042 0.044 0.051 0.046 0.046 0.055 0.046 0.047 0.058 p Value 2.7E−7 3.6E−5 0.014 0.0095 0.019 0.050 0.012 0.052 0.10 nCohort 49 51 103 70 70 111 79 79 116 Non-persistent nCohort 101 99 46 80 80 38 71 71 33 Persistent Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 85% 84% 87% 82% 81% 87% 82% 80% 85% Specificity 47% 43% 30% 34% 33% 29% 32% 30% 28% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 59% 59% 63% 55% 55% 58% 56% 55% 58% Specificity 69% 67% 55% 56% 56% 52% 56% 54% 52% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 33% 31% 35% 31% 31% 37% 34% 32% 36% Specificity 90% 86% 79% 81% 81% 78% 82% 81% 78% OR Quartile 2 5.07 3.94 2.87 2.46 2.12 2.67 2.07 1.78 2.13 p Value 5.0E−5 4.9E−4 0.031 0.020 0.050 0.060 0.063 0.14 0.15 Lower limit 2.31 1.82 1.10 1.15 1.00 0.958 0.960 0.834 0.758 of 95% CI Upper limit 11.1 8.50 7.47 5.25 4.49 7.46 4.44 3.78 6.01 of 95% CI OR Quartile 3 3.32 2.83 2.11 1.54 1.54 1.50 1.62 1.46 1.45 p Value 0.0012 0.0039 0.040 0.19 0.19 0.28 0.14 0.25 0.35 Lower limit 1.61 1.40 1.04 0.806 0.806 0.715 0.850 0.765 0.666 of 95% CI Upper limit 6.85 5.73 4.32 2.93 2.93 3.17 3.09 2.77 3.17 of 95% CI OR Quartile 4 4.27 2.87 1.96 1.99 1.99 2.11 2.37 2.04 1.98 p Value 0.0050 0.022 0.085 0.078 0.078 0.066 0.026 0.062 0.11 Lower limit 1.55 1.16 0.911 0.927 0.927 0.951 1.11 0.965 0.860 of 95% CI Upper limit 11.8 7.07 4.23 4.29 4.29 4.70 5.06 4.33 4.55 of 95% CI

TABLE 19.8 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 67.1 119 85.2 113 88.6 113 Average 81.4 129 97.7 127 98.2 128 Stdev 41.9 69.9 52.9 72.9 51.4 75.0 p (t-test) 2.3E−5 0.0071 0.0046 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 174 401 246 401 246 401 n (Patient) 49 101 69 81 75 75 sCr only Median 73.7 118 87.0 113 91.4 113 Average 85.8 127 98.5 126 98.9 128 Stdev 43.9 70.8 52.4 73.4 51.0 75.6 p (t-test) 1.9E−4 0.011 0.0073 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 182 401 246 401 246 401 n (Patient) 51 99 69 81 75 75 UO only Median 92.8 122 98.9 112 99.7 111 Average 103 134 106 133 107 134 Stdev 58.8 74.4 57.8 79.7 57.1 83.9 p (t-test) 0.0073 0.022 0.028 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 274 401 274 401 274 401 n (Patient) 98 51 105 44 110 39 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.68 0.63 0.62 0.61 0.60 0.62 0.60 0.58 SE 0.042 0.044 0.049 0.046 0.046 0.052 0.046 0.046 0.054 p Value 2.7E−7 3.6E−5 0.0086 0.0099 0.020 0.065 0.012 0.023 0.13 nCohort 49 51 98 69 69 105 75 75 110 Non-persistent nCohort 101 99 51 81 81 44 75 75 39 Persistent Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 85% 84% 88% 83% 81% 86% 83% 81% 85% Specificity 47% 43% 32% 35% 33% 30% 33% 32% 28% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 59% 59% 61% 54% 54% 55% 55% 55% 54% Specificity 69% 67% 55% 55% 55% 51% 55% 55% 51% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 33% 31% 33% 31% 31% 34% 32% 32% 33% Specificity 90% 86% 79% 81% 81% 78% 81% 81% 77% OR Quartile 2 5.07 3.94 3.47 2.55 2.20 2.65 2.38 2.05 2.16 p Value 5.0E−5 4.9E−4 0.010 0.016 0.040 0.046 0.026 0.063 0.12 Lower limit 2.31 1.82 1.34 1.19 1.04 1.02 1.11 0.962 0.823 of 95% CI Upper limit 11.1 8.50 8.99 5.46 4.66 6.91 5.13 4.37 5.66 of 95% CI OR Quartile 3 3.32 2.83 1.90 1.46 1.46 1.27 1.45 1.45 1.21 p Value 0.0012 0.0039 0.067 0.25 0.25 0.51 0.25 0.25 0.61 Lower limit 1.61 1.40 0.955 0.765 0.765 0.627 0.765 0.765 0.582 of 95% CI Upper limit 6.85 5.73 3.79 2.78 2.78 2.57 2.77 2.77 2.52 of 95% CI OR Quartile 4 4.27 2.87 1.83 1.92 1.92 1.84 2.05 2.05 1.70 p Value 0.0050 0.022 0.12 0.094 0.094 0.12 0.063 0.063 0.19 Lower limit 1.55 1.16 0.861 0.894 0.894 0.849 0.962 0.962 0.763 of 95% CI Upper limit 11.8 7.07 3.91 4.14 4.14 4.01 4.37 4.37 3.79 of 95% CI

TABLE 19.9 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 67.1 119 84.6 115 87.8 115 Average 81.4 129 95.8 128 96.4 130 Stdev 41.9 69.9 50.8 73.3 49.4 75.4 p (t-test) 2.3E−5 0.0028 0.0018 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 174 401 246 401 246 401 n (Patient) 49 101 68 82 74 76 sCr only Median 73.7 118 86.1 115 90.0 115 Average 85.8 127 96.6 127 97.2 129 Stdev 43.9 70.8 50.4 73.9 49.0 76.0 p (t-test) 1.9E−4 0.0045 0.0029 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 182 401 246 401 246 401 n (Patient) 51 99 68 82 74 76 UO only Median 91.4 120 95.2 113 96.1 112 Average 102 133 105 132 106 133 Stdev 58.2 74.3 57.6 77.6 56.9 81.2 p (t-test) 0.0056 0.015 0.020 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 274 401 274 401 274 401 n (Patient) 95 54 100 49 105 44 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.68 0.63 0.63 0.62 0.60 0.63 0.62 0.59 SE 0.042 0.044 0.048 0.045 0.046 0.050 0.045 0.046 0.052 p Value 2.7E−7 3.6E−5 0.0069 0.0042 0.0091 0.039 0.0052 0.011 0.085 nCohort 49 51 95 68 68 100 74 74 105 Non-persistent nCohort 101 99 54 82 82 49 76 76 44 Persistent Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 85% 84% 87% 83% 82% 86% 83% 82% 84% Specificity 47% 43% 32% 35% 34% 30% 34% 32% 29% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 59% 59% 61% 55% 55% 57% 55% 55% 57% Specificity 69% 67% 56% 56% 56% 53% 55% 55% 52% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 33% 31% 33% 32% 32% 33% 33% 33% 32% Specificity 90% 86% 79% 82% 82% 78% 82% 82% 77% OR Quartile 2 5.07 3.94 3.10 2.65 2.28 2.57 2.47 2.13 2.11 p Value 5.0E−5 4.9E−4 0.014 0.012 0.031 0.041 0.021 0.051 0.11 Lower limit 2.31 1.82 1.25 1.24 1.08 1.04 1.15 0.997 0.849 of 95% CI Upper limit 11.1 8.50 7.65 5.67 4.84 6.37 5.32 4.53 5.26 of 95% CI OR Quartile 3 3.32 2.83 1.98 1.54 1.54 1.50 1.53 1.53 1.45 p Value 0.0012 0.0039 0.049 0.19 0.19 0.25 0.19 0.19 0.31 Lower limit 1.61 1.40 1.00 0.807 0.807 0.755 0.806 0.806 0.712 of 95% CI Upper limit 6.85 5.73 3.92 2.94 2.94 2.99 2.92 2.92 2.94 of 95% CI OR Quartile 4 4.27 2.87 1.88 2.17 2.17 1.72 2.30 2.30 1.58 p Value 0.0050 0.022 0.10 0.051 0.051 0.16 0.033 0.033 0.25 Lower limit 1.55 1.16 0.885 0.995 0.995 0.802 1.07 1.07 0.721 of 95% CI Upper limit 11.8 7.07 3.97 4.72 4.72 3.68 4.95 4.95 3.44 of 95% CI

TABLE 19.10 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 67.1 118 84.0 118 86.1 118 Average 82.3 127 94.5 128 95.0 129 Stdev 42.4 69.9 50.0 72.9 48.9 74.4 p (t-test) 7.1E−5 0.0020 0.0013 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 174 401 246 401 246 401 n (Patient) 47 103 65 85 70 80 sCr only Median 71.0 117 86.1 115 90.0 115 Average 86.0 127 95.6 127 96.3 129 Stdev 44.4 70.6 50.0 73.4 48.7 75.4 p (t-test) 2.6E−4 0.0033 0.0022 Min 23.6 13.2 16.4 13.2 16.4 13.2 Max 182 401 246 401 246 401 n (Patient) 50 100 66 84 72 78 UO only Median 90.0 122 94.2 118 94.3 115 Average 101 135 102 134 104 134 Stdev 55.1 76.3 55.1 78.2 54.7 80.7 p (t-test) 0.0019 0.0046 0.0079 Min 16.4 13.2 16.4 13.2 16.4 13.2 Max 260 401 260 401 260 401 n (Patient) 92 57 95 54 99 50 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.68 0.64 0.64 0.62 0.62 0.64 0.62 0.60 SE 0.043 0.044 0.048 0.045 0.045 0.049 0.045 0.045 0.050 p Value 2.2E−6 5.3E−5 0.0036 0.0024 0.0058 0.014 0.0025 0.0073 0.038 nCohort Non-persistent 47 50 92 65 66 95 70 72 99 nCohort Persistent 103 100 57 85 84 54 80 78 50 Cutoff Quartile 2 63.3 63.3 66.7 63.3 63.3 66.7 63.3 63.3 66.7 Sensitivity 84% 84% 86% 82% 82% 85% 82% 82% 84% Specificity 47% 44% 32% 35% 35% 31% 34% 33% 29% Cutoff Quartile 3 101 101 101 101 101 101 101 101 101 Sensitivity 58% 58% 61% 55% 55% 59% 56% 55% 58% Specificity 68% 66% 57% 57% 56% 55% 57% 56% 54% Cutoff Quartile 4 142 142 142 142 142 142 142 142 142 Sensitivity 32% 31% 35% 33% 32% 35% 34% 33% 34% Specificity 89% 86% 80% 85% 83% 80% 84% 83% 79% OR Quartile 2 4.78 4.12 2.82 2.56 2.46 2.53 2.46 2.29 2.17 p Value 8.8E−5 3.3E−4 0.019 0.015 0.019 0.036 0.020 0.033 0.080 Lower limit of 95% CI 2.19 1.90 1.18 1.20 1.16 1.06 1.15 1.07 0.910 Upper limit of 95% CI 10.5 8.94 6.71 5.44 5.23 6.02 5.25 4.88 5.20 OR Quartile 3 2.98 2.68 2.07 1.63 1.54 1.76 1.71 1.54 1.59 p Value 0.0033 0.0063 0.035 0.14 0.19 0.10 0.10 0.19 0.18 Lower limit of 95% CI 1.44 1.32 1.05 0.852 0.807 0.894 0.897 0.806 0.801 Upper limit of 95% CI 6.16 5.44 4.06 3.13 2.95 3.46 3.28 2.93 3.16 OR Quartile 4 3.96 2.76 2.22 2.70 2.37 2.17 2.73 2.50 1.91 p Value 0.0079 0.028 0.037 0.016 0.033 0.043 0.013 0.021 0.093 Lower limit of 95% CI 1.43 1.12 1.05 1.20 1.07 1.02 1.24 1.15 0.897 Upper limit of 95% CI 10.9 6.82 4.70 6.08 5.23 4.60 6.04 5.44 4.08

Example 20. Use of Tenascin for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Tenascin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 20.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 47000 59300 47500 58200 48100 58800 Average 51400 61900 52200 62100 52800 62200 Stdev 24600 27900 24700 28500 25000 28700 p (t-test) 0.018 0.031 0.051 Min 17100 15600 17100 15600 17100 15600 Max 140000 141000 140000 141000 140000 141000 n (Patient) 95 55 103 47 110 40 sCr only Median 45800 59600 47500 59300 48100 58800 Average 51200 62200 52200 61900 53100 60900 Stdev 24700 27700 25000 28000 25400 28000 p (t-test) 0.013 0.035 0.10 Min 17100 15600 17100 15600 17100 15600 Max 140000 141000 140000 141000 140000 141000 n (Patient) 94 56 103 47 108 42 UO only Median 51300 62000 51300 62000 51700 61000 Average 53600 65600 53600 65600 53800 65100 Stdev 24700 34600 24700 34600 24600 35500 p (t-test) 0.062 0.062 0.085 Min 17100 15600 17100 15600 17100 15600 Max 140000 141000 140000 141000 140000 141000 n (Patient) 130 19 130 19 131 18 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.62 0.62 0.60 0.61 0.61 0.60 0.60 0.59 0.59 SE 0.048 0.048 0.073 0.051 0.051 0.073 0.054 0.053 0.075 p Value 0.015 0.0093 0.17 0.038 0.033 0.17 0.063 0.10 0.24 nCohort Non-persistent 95 94 130 103 103 130 110 108 131 nCohort Persistent 55 56 19 47 47 19 40 42 18 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 80% 80% 79% 81% 81% 79% 82% 81% 78% Specificity 28% 29% 25% 28% 28% 25% 28% 28% 25% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 60% 61% 63% 60% 62% 63% 60% 60% 61% Specificity 56% 56% 52% 54% 55% 52% 54% 54% 51% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 33% 34% 37% 34% 34% 37% 32% 31% 33% Specificity 79% 80% 76% 79% 79% 76% 77% 77% 76% OR Quartile 2 1.59 1.65 1.28 1.65 1.65 1.28 1.85 1.63 1.18 p Value 0.26 0.22 0.68 0.24 0.24 0.68 0.19 0.27 0.78 Lower limit of 95% CI 0.716 0.743 0.395 0.711 0.711 0.395 0.741 0.680 0.362 Upper limit of 95% CI 3.52 3.66 4.12 3.85 3.85 4.12 4.62 3.93 3.83 OR Quartile 3 1.89 2.00 1.82 1.76 2.00 1.82 1.74 1.71 1.65 p Value 0.064 0.044 0.24 0.11 0.055 0.24 0.14 0.15 0.33 Lower limit of 95% CI 0.964 1.02 0.675 0.872 0.987 0.675 0.832 0.828 0.601 Upper limit of 95% CI 3.72 3.92 4.92 3.54 4.04 4.92 3.62 3.52 4.51 OR Quartile 4 1.82 2.03 1.86 1.90 1.90 1.86 1.64 1.49 1.55 p Value 0.12 0.064 0.23 0.10 0.10 0.23 0.23 0.33 0.42 Lower limit of 95% CI 0.863 0.959 0.675 0.884 0.884 0.675 0.737 0.674 0.537 Upper limit of 95% CI 3.86 4.28 5.14 4.09 4.09 5.14 3.64 3.29 4.46

TABLE 20.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 45800 58800 47200 58800 47500 59300 Average 49800 63100 50400 63500 51200 63700 Stdev 22700 29100 22900 29500 23400 29900 p (t-test) 0.0021 0.0027 0.0058 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 88 62 94 56 101 49 sCr only Median 45800 58800 47500 58200 47900 58200 Average 50500 62000 51100 62000 52200 61300 Stdev 24500 27400 24700 27600 25000 27800 p (t-test) 0.0080 0.013 0.043 Min 17100 15600 17100 15600 17100 15600 Max 140000 141000 140000 141000 140000 141000 n (Patient) 88 62 93 57 99 51 UO only Median 50800 61000 50800 61000 51300 59900 Average 52600 67100 52600 67100 52800 66800 Stdev 23600 34500 23600 34500 23600 35200 p (t-test) 0.010 0.010 0.014 Min 17100 15600 17100 15600 17100 15600 Max 129000 141000 129000 141000 129000 141000 n (Patient) 123 26 123 26 124 25 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.63 0.63 0.62 0.63 0.62 0.62 0.62 0.60 0.61 SE 0.047 0.047 0.063 0.048 0.048 0.063 0.050 0.050 0.065 p Value 0.0040 0.0059 0.054 0.0056 0.011 0.054 0.013 0.042 0.082 nCohort Non-persistent 88 88 123 94 93 123 101 99 124 nCohort Persistent 62 62 26 56 57 26 49 51 25 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 81% 81% 81% 80% 81% 81% 82% 80% 80% Specificity 30% 30% 26% 29% 29% 26% 29% 28% 26% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 60% 60% 62% 61% 60% 62% 59% 57% 60% Specificity 57% 57% 52% 56% 56% 52% 54% 54% 52% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 34% 34% 38% 36% 35% 38% 35% 33% 36% Specificity 81% 81% 77% 81% 81% 77% 79% 79% 77% OR Quartile 2 1.75 1.75 1.48 1.65 1.71 1.48 1.79 1.62 1.39 p Value 0.16 0.16 0.47 0.22 0.19 0.47 0.18 0.25 0.54 Lower limit of 95% CI 0.802 0.802 0.514 0.743 0.772 0.514 0.771 0.714 0.482 Upper limit of 95% CI 3.81 3.81 4.24 3.66 3.79 4.24 4.15 3.66 4.01 OR Quartile 3 1.95 1.95 1.74 2.00 1.87 1.74 1.73 1.52 1.60 p Value 0.048 0.048 0.21 0.044 0.066 0.21 0.12 0.23 0.29 Lower limit of 95% CI 1.01 1.01 0.730 1.02 0.960 0.730 0.868 0.769 0.667 Upper limit of 95% CI 3.77 3.77 4.12 3.92 3.66 4.12 3.46 3.00 3.84 OR Quartile 4 2.14 2.14 2.12 2.35 2.25 2.12 2.02 1.86 1.84 p Value 0.046 0.046 0.100 0.026 0.034 0.100 0.069 0.11 0.19 Lower limit of 95% CI 1.01 1.01 0.866 1.11 1.07 0.866 0.947 0.872 0.737 Upper limit of 95% CI 4.51 4.51 5.19 4.97 4.76 5.19 4.33 3.95 4.61

TABLE 20.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 43800 59900 44100 60600 45800 61000 Average 48000 64300 48000 65100 48900 66000 Stdev 21700 28700 21500 28900 21800 29700 p (t-test) 1.1E−4 5.3E−5 8.3E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 83 67 86 64 94 56 sCr only Median 43700 60600 44100 60600 47000 59900 Average 47600 64500 48200 64800 49300 64900 Stdev 21600 28500 22000 28700 22200 29600 p (t-test) 6.7E−5 9.8E−5 3.3E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 82 68 86 64 93 57 UO only Median 47500 64800 47500 64800 47600 64300 Average 50300 71300 50300 71300 50600 71200 Stdev 22200 32500 22200 32500 22200 33000 p (t-test) 2.9E−5 2.9E−5 4.7E−5 Min 17100 15600 17100 15600 17100 15600 Max 129000 141000 129000 141000 129000 141000 n (Patient) 115 34 115 34 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.68 0.70 0.68 0.68 0.70 0.67 0.66 0.69 SE 0.045 0.044 0.055 0.045 0.045 0.055 0.047 0.047 0.056 p Value 1.3E−4 5.3E−5 3.5E−4 6.4E−5 8.9E−5 3.5E−4 2.2E−4 7.5E−4 7.0E−4 nCohort 83 82 115 86 86 115 94 93 116 Non-persistent nCohort Persistent 67 68 34 64 64 34 56 57 33 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 82% 82% 85% 83% 83% 85% 84% 82% 85% Specificity 31% 32% 28% 31% 31% 28% 31% 30% 28% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 63% 63% 71% 64% 64% 71% 62% 61% 70% Specificity 60% 61% 56% 60% 60% 56% 57% 57% 55% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 37% 38% 47% 39% 39% 47% 39% 39% 45% Specificity 84% 85% 81% 85% 85% 81% 83% 83% 80% OR Quartile 2 2.09 2.17 2.24 2.20 2.20 2.24 2.33 2.02 2.13 p Value 0.063 0.051 0.13 0.051 0.051 0.13 0.048 0.089 0.15 Lower limit of 0.960 0.995 0.796 0.998 0.998 0.796 1.01 0.897 0.758 95% CI Upper limit of 4.55 4.72 6.28 4.87 4.87 6.28 5.38 4.57 6.01 95% CI OR Quartile 3 2.55 2.69 3.01 2.73 2.73 3.01 2.25 2.11 2.83 p Value 0.0057 0.0035 0.0088 0.0033 0.0033 0.0088 0.019 0.030 0.014 Lower limit of 1.31 1.38 1.32 1.40 1.40 1.32 1.14 1.08 1.24 95% CI Upper limit of 4.94 5.22 6.87 5.32 5.32 6.87 4.43 4.13 6.48 95% CI OR Quartile 4 3.21 3.61 3.76 3.60 3.60 3.76 3.15 3.02 3.37 p Value 0.0031 0.0013 0.0015 0.0012 0.0012 0.0015 0.0030 0.0042 0.0038 Lower limit of 1.48 1.65 1.66 1.66 1.66 1.66 1.48 1.42 1.48 95% CI Upper limit of 6.93 7.91 8.52 7.81 7.81 8.52 6.74 6.45 7.68 95% CI

TABLE 20.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 43800 59900 44300 59900 47000 59900 Average 48000 63800 48200 64500 48800 65200 Stdev 21800 28600 21500 29100 21300 30000 p (t-test) 1.9E−4 1.2E−4 1.4E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 81 69 85 65 91 59 sCr only Median 43800 59900 44100 60600 45800 61000 Average 47900 63900 47900 64700 48500 65400 Stdev 21600 28600 21500 28900 21200 29800 p (t-test) 1.5E−4 7.2E−5 8.4E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 81 69 84 66 90 60 UO only Median 47500 64800 47500 64300 47500 63200 Average 49600 70200 49700 70400 50000 70200 Stdev 21100 32800 21000 33200 21100 33600 p (t-test) 1.3E−5 1.6E−5 2.6E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 109 40 110 39 111 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.67 0.69 0.67 0.68 0.68 0.66 0.67 0.68 SE 0.045 0.045 0.052 0.045 0.045 0.052 0.046 0.046 0.053 p Value 2.0E−4 1.6E−4 2.6E−4 1.9E−4 8.3E−5 4.4E−4 5.3E−4 2.5E−4 8.6E−4 nCohort 81 81 109 85 84 110 91 90 111 Non-persistent nCohort Persistent 69 69 40 65 66 39 59 60 38 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 81% 81% 82% 82% 82% 82% 81% 82% 82% Specificity 31% 31% 28% 31% 31% 27% 30% 30% 27% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 62% 62% 70% 63% 64% 69% 61% 62% 68% Specificity 60% 60% 57% 60% 61% 56% 57% 58% 56% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 36% 38% 45% 38% 39% 46% 39% 40% 45% Specificity 84% 85% 82% 85% 86% 82% 84% 84% 81% OR Quartile 2 1.92 1.92 1.79 1.95 2.02 1.71 1.84 1.91 1.64 p Value 0.094 0.094 0.21 0.093 0.077 0.25 0.13 0.11 0.29 Lower limit 0.894 0.894 0.715 0.894 0.927 0.684 0.832 0.863 0.653 of 95% CI Upper limit of 4.14 4.14 4.48 4.24 4.39 4.30 4.08 4.22 4.12 95% CI OR Quartile 3 2.53 2.53 3.08 2.56 2.70 2.91 2.09 2.20 2.74 p Value 0.0058 0.0058 0.0045 0.0055 0.0034 0.0072 0.031 0.021 0.011 Lower limit of 1.31 1.31 1.42 1.32 1.39 1.34 1.07 1.13 1.26 95% CI Upper limit of 4.90 4.90 6.68 4.98 5.26 6.32 4.07 4.29 5.98 95% CI OR Quartile 4 2.97 3.48 3.64 3.46 3.90 3.86 3.24 3.62 3.47 p Value 0.0056 0.0018 0.0013 0.0017 6.9E−4 8.7E−4 0.0025 0.0010 0.0022 Lower limit of 1.38 1.59 1.65 1.60 1.78 1.74 1.51 1.68 1.56 95% CI Upper limit of 6.42 7.61 8.02 7.50 8.56 8.54 6.93 7.81 7.69 95% CI

TABLE 20.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 43200 60600 43500 61300 44500 62800 Average 46900 64300 46900 65100 47700 66000 Stdev 21100 28400 20900 28600 20600 29700 p (t-test) 3.3E−5 1.5E−5 1.8E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 78 72 81 69 88 62 sCr only Median 43500 59900 43700 60600 44500 61000 Average 47100 64300 47200 65100 47900 65800 Stdev 21000 28600 20900 28800 20700 29700 p (t-test) 4.4E−5 2.0E−5 2.5E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 79 71 82 68 88 62 UO only Median 47400 65200 47400 65200 47500 64800 Average 49400 70300 49400 70300 49600 70200 Stdev 21000 32400 21000 32400 21100 32800 p (t-test) 7.6E−6 7.6E−6 1.3E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 108 41 108 41 109 40 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.68 0.70 0.69 0.69 0.70 0.68 0.68 0.69 SE 0.043 0.044 0.051 0.044 0.044 0.051 0.045 0.045 0.052 p Value 1.9E−5 3.8E−5 1.2E−4 9.2E−6 1.9E−5 1.2E−4 4.0E−5 7.0E−5 2.5E−4 nCohort 78 79 108 81 82 108 88 88 109 Non-persistent nCohort Persistent 72 71 41 69 68 41 62 62 40 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 82% 82% 83% 83% 82% 83% 82% 82% 82% Specificity 32% 32% 28% 32% 32% 28% 31% 31% 28% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 64% 63% 71% 65% 65% 71% 63% 63% 70% Specificity 63% 62% 57% 63% 62% 57% 59% 59% 57% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 38% 38% 46% 39% 40% 46% 40% 40% 45% Specificity 86% 86% 82% 86% 87% 82% 85% 85% 82% OR Quartile 2 2.14 2.07 1.87 2.25 2.17 1.87 2.05 2.05 1.79 p Value 0.051 0.063 0.18 0.042 0.051 0.18 0.076 0.076 0.21 Lower limit of 95% CI 0.995 0.960 0.747 1.03 0.995 0.747 0.928 0.928 0.715 Upper limit of 95% CI 4.61 4.44 4.67 4.89 4.72 4.67 4.54 4.54 4.48 OR Quartile 3 2.99 2.83 3.26 3.19 3.02 3.26 2.45 2.45 3.08 p Value 0.0012 0.0021 0.0028 7.0E−4 0.0012 0.0028 0.0086 0.0086 0.0045 Lower limit of 95% CI 1.54 1.46 1.50 1.63 1.55 1.50 1.26 1.26 1.42 Upper limit of 95% CI 5.81 5.49 7.06 6.23 5.88 7.06 4.78 4.78 6.68 OR Quartile 4 3.65 3.79 4.05 4.09 4.25 4.05 3.90 3.90 3.64 p Value 0.0014 0.0010 5.1E−4 5.5E−4 3.9E−4 5.1E−4 6.0E−4 6.0E−4 0.0013 Lower limit of 95% CI 1.65 1.71 1.84 1.84 1.91 1.84 1.79 1.79 1.65 Upper limit of 95% CI 8.10 8.42 8.90 9.09 9.46 8.90 8.48 8.48 8.02

Example 21. Use of Tenascin for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Tenascin is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 21.1 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 37500 57200 43800 58200 47000 58200 Average 44000 60900 49500 61000 50100 62000 Stdev 21400 26800 25000 26400 23900 27800 p (t-test) 1.6E−4 0.0070 0.0058 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 140000 141000 140000 141000 n (Patient) 50 100 75 75 85 65 sCr only Median 37500 57200 43500 59300 45800 58800 Average 44300 61100 47300 62800 49700 62800 Stdev 21500 26800 22200 27700 22800 28800 p (t-test) 1.5E−4 2.3E−4 0.0024 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 52 98 73 77 86 64 UO only Median 47800 62000 48300 62000 48400 63200 Average 52400 63400 52900 64300 52900 66700 Stdev 24900 28800 24700 30900 24400 32800 p (t-test) 0.027 0.036 0.018 Min 17100 15600 17100 15600 17100 15600 Max 140000 141000 140000 141000 140000 141000 n (Patient) 112 37 120 29 125 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.70 0.62 0.64 0.67 0.61 0.63 0.63 0.63 SE 0.043 0.043 0.055 0.045 0.044 0.061 0.046 0.046 0.065 p Value 2.9E−6 3.8E−6 0.024 0.0021 8.0E−5 0.063 0.0049 0.0039 0.050 nCohort Non-persistent 50 52 112 75 73 120 85 86 125 nCohort Persistent 100 98 37 75 77 29 65 64 24 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 81% 82% 81% 81% 82% 79% 80% 81% 79% Specificity 38% 38% 27% 32% 33% 26% 29% 30% 26% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 59% 59% 68% 60% 62% 66% 60% 61% 67% Specificity 68% 67% 55% 60% 63% 53% 58% 58% 53% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 32% 33% 35% 32% 35% 34% 35% 36% 38% Specificity 88% 88% 78% 81% 85% 77% 82% 83% 77% OR Quartile 2 2.61 2.78 1.57 2.05 2.20 1.34 1.67 1.88 1.31 p Value 0.013 0.0082 0.34 0.063 0.041 0.57 0.19 0.11 0.62 Lower limit of 95% CI 1.22 1.30 0.623 0.962 1.03 0.498 0.775 0.862 0.451 Upper limit of 95% CI 5.58 5.92 3.95 4.37 4.70 3.58 3.59 4.09 3.79 OR Quartile 3 3.06 2.99 2.58 2.25 2.82 2.17 2.04 2.17 2.24 p Value 0.0022 0.0024 0.017 0.015 0.0021 0.072 0.033 0.022 0.086 Lower limit of 95% CI 1.50 1.47 1.18 1.17 1.45 0.932 1.06 1.12 0.893 Upper limit of 95% CI 6.25 6.05 5.65 4.32 5.47 5.06 3.94 4.19 5.61 OR Quartile 4 3.45 3.72 1.88 2.05 3.04 1.73 2.56 2.66 1.99 p Value 0.011 0.0067 0.12 0.063 0.0060 0.22 0.015 0.011 0.15 Lower limit of 95% CI 1.33 1.44 0.840 0.962 1.38 0.721 1.20 1.25 0.788 Upper limit of 95% CI 8.93 9.61 4.23 4.37 6.73 4.15 5.44 5.65 5.01

TABLE 21.2 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 37400 58200 42700 59100 43800 59300 Average 43600 60900 47100 62400 48000 63300 Stdev 21400 26600 22700 27200 21900 28500 p (t-test) 1.1E−4 2.9E−4 3.0E−4 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 49 101 70 80 79 71 sCr only Median 37400 58200 42700 59100 44300 58200 Average 43900 61100 47000 62500 48700 62600 Stdev 21500 26700 22500 27300 22700 28100 p (t-test) 9.9E−5 2.3E−4 0.0011 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 51 99 70 80 79 71 UO only Median 47500 64000 47700 64000 47800 64600 Average 49900 66900 50600 68300 50700 70600 Stdev 22100 31000 22100 32800 21900 34100 p (t-test) 2.1E−4 2.9E−4 8.9E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 103 46 111 38 116 33 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.71 0.67 0.68 0.68 0.66 0.66 0.65 0.68 SE 0.043 0.042 0.050 0.043 0.043 0.054 0.045 0.045 0.056 p Value 8.2E−7 1.1E−6 6.5E−4 5.6E−5 4.4E−5 0.0025 2.5E−4 9.4E−4 0.0017 nCohort 49 51 103 70 70 111 79 79 116 Non-persistent nCohort Persistent 101 99 46 80 80 38 71 71 33 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 81% 82% 83% 82% 82% 82% 82% 82% 82% Specificity 39% 39% 28% 34% 34% 27% 32% 32% 27% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 59% 60% 67% 62% 62% 66% 62% 61% 67% Specificity 69% 69% 57% 64% 64% 55% 61% 59% 54% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 32% 32% 39% 32% 34% 39% 35% 35% 42% Specificity 88% 88% 81% 83% 84% 79% 84% 84% 79% OR Quartile 2 2.73 2.90 1.86 2.46 2.46 1.64 2.07 2.07 1.64 p Value 0.0096 0.0060 0.16 0.020 0.020 0.29 0.063 0.063 0.32 Lower limit of 1.28 1.36 0.776 1.15 1.15 0.653 0.960 0.960 0.619 95% CI Upper limit of 5.85 6.20 4.47 5.25 5.25 4.12 4.44 4.44 4.35 95% CI OR Quartile 3 3.32 3.23 2.77 3.00 3.00 2.35 2.52 2.26 2.38 p Value 0.0012 0.0013 0.0062 0.0012 0.0012 0.029 0.0059 0.015 0.036 Lower limit of 1.61 1.58 1.34 1.54 1.54 1.09 1.31 1.17 1.06 95% CI Upper limit of 6.85 6.59 5.75 5.84 5.84 5.05 4.87 4.34 5.35 95% CI OR Quartile 4 3.32 3.58 2.67 2.33 2.73 2.50 2.76 2.76 2.82 p Value 0.013 0.0085 0.012 0.033 0.013 0.024 0.0097 0.0097 0.013 Lower limit of 1.28 1.38 1.24 1.07 1.24 1.13 1.28 1.28 1.24 95% CI Upper limit of 8.61 9.26 5.75 5.07 6.04 5.53 5.95 5.95 6.44 95% CI

TABLE 21.3 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 37400 58200 42700 59300 43500 59300 Average 43600 60900 46600 62600 47200 63300 Stdev 21400 26600 22500 27100 22000 27900 p (t-test) 1.1E−4 1.5E−4 1.4E−4 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 49 101 69 81 75 75 sCr only Median 37400 58200 42700 59300 43500 59300 Average 43900 61100 46500 62700 47100 63400 Stdev 21500 26700 22300 27200 21800 27900 p (t-test) 9.9E−5 1.2E−4 1.0E−4 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 51 99 69 81 75 75 UO only Median 47200 64300 47500 64000 47500 64600 Average 48600 67800 49400 68800 49600 70800 Stdev 21200 30500 21400 31700 21200 32700 p (t-test) 1.4E−5 2.7E−5 7.9E−6 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 98 51 105 44 110 39 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.71 0.69 0.68 0.69 0.68 0.68 0.68 0.70 SE 0.043 0.042 0.047 0.043 0.043 0.050 0.044 0.044 0.052 p Value 8.2E−7 1.1E−6 3.9E−5 1.8E−5 1.3E−5 2.4E−4 4.8E−5 3.7E−5 1.5E−4 nCohort 49 51 98 69 69 105 75 75 110 Non-persistent nCohort Persistent 101 99 51 81 81 44 75 75 39 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 81% 82% 84% 83% 83% 84% 83% 83% 85% Specificity 39% 39% 30% 35% 35% 29% 33% 33% 28% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 59% 60% 69% 63% 63% 68% 63% 63% 69% Specificity 69% 69% 59% 65% 65% 57% 63% 63% 56% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 32% 32% 41% 33% 35% 41% 36% 37% 44% Specificity 88% 88% 83% 84% 86% 81% 85% 87% 81% OR Quartile 2 2.73 2.90 2.26 2.55 2.55 2.11 2.38 2.38 2.16 p Value 0.0096 0.0060 0.067 0.016 0.016 0.11 0.026 0.026 0.12 Lower limit of 95% CI 1.28 1.36 0.946 1.19 1.19 0.849 1.11 1.11 0.823 Upper limit of 95% CI 5.85 6.20 5.39 5.46 5.46 5.26 5.13 5.13 5.66 OR Quartile 3 3.32 3.23 3.17 3.19 3.19 2.86 2.82 2.82 2.91 p Value 0.0012 0.0013 0.0016 7.0E−4 7.0E−4 0.0056 0.0022 0.0022 0.0072 Lower limit of 95% CI 1.61 1.58 1.55 1.63 1.63 1.36 1.45 1.45 1.34 Upper limit of 95% CI 6.85 6.59 6.49 6.23 6.23 6.01 5.46 5.46 6.32 OR Quartile 4 3.32 3.58 3.34 2.64 3.12 2.94 3.27 3.87 3.27 p Value 0.013 0.0085 0.0020 0.017 0.0061 0.0063 0.0035 0.0011 0.0033 Lower limit of 95% CI 1.28 1.38 1.55 1.19 1.38 1.36 1.48 1.72 1.48 Upper limit of 95% CI 8.61 9.26 7.16 5.83 7.02 6.38 7.25 8.74 7.23

TABLE 21.4 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 37400 58200 42700 59100 43700 59100 Average 43600 60900 46900 62200 47500 62800 Stdev 21400 26600 22500 27300 22000 28000 p (t-test) 1.1E−4 3.1E−4 2.8E−4 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 49 101 68 82 74 76 sCr only Median 37400 58200 42700 59100 43700 59100 Average 43900 61100 46800 62300 47400 63000 Stdev 21500 26700 22400 27300 21800 28000 p (t-test) 9.9E−5 2.4E−4 2.2E−4 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 51 99 68 82 74 76 UO only Median 47500 62800 47500 62000 47500 63200 Average 49200 65600 49700 66200 49800 67800 Stdev 21200 31000 21400 31600 21200 32600 p (t-test) 1.9E−4 2.5E−4 1.1E−4 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 95 54 100 49 105 44 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.71 0.66 0.67 0.68 0.65 0.67 0.67 0.66 SE 0.043 0.042 0.048 0.043 0.043 0.049 0.044 0.044 0.051 p Value 8.2E−7 1.1E−6 8.0E−4 5.6E−5 4.4E−5 0.0019 1.4E−4 1.1E−4 0.0015 nCohort 49 51 95 68 68 100 74 74 105 Non-persistent nCohort Persistent 101 99 54 82 82 49 76 76 44 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 81% 82% 80% 82% 82% 80% 82% 82% 80% Specificity 39% 39% 27% 34% 34% 27% 32% 32% 27% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 59% 60% 65% 62% 62% 65% 62% 62% 66% Specificity 69% 69% 58% 65% 65% 57% 62% 62% 56% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 32% 32% 39% 33% 34% 39% 36% 37% 41% Specificity 88% 88% 82% 84% 85% 81% 85% 86% 81% OR Quartile 2 2.73 2.90 1.47 2.28 2.28 1.44 2.13 2.13 1.41 p Value 0.0096 0.0060 0.34 0.031 0.031 0.38 0.051 0.051 0.42 Lower limit of 1.28 1.36 0.661 1.08 1.08 0.633 0.997 0.997 0.604 95% CI Upper limit of 5.85 6.20 3.28 4.84 4.84 3.29 4.53 4.53 3.31 95% CI OR Quartile 3 3.32 3.23 2.53 3.02 3.02 2.50 2.66 2.66 2.48 p Value 0.0012 0.0013 0.0084 0.0012 0.0012 0.011 0.0036 0.0036 0.015 Lower limit of 1.61 1.58 1.27 1.55 1.55 1.23 1.38 1.38 1.19 95% CI Upper limit of 6.85 6.59 5.06 5.88 5.88 5.07 5.15 5.15 5.16 95% CI OR Quartile 4 3.32 3.58 2.92 2.54 3.01 2.70 3.16 3.73 2.94 p Value 0.013 0.0085 0.0056 0.021 0.0078 0.011 0.0046 0.0015 0.0063 Lower limit of 1.28 1.38 1.37 1.15 1.34 1.26 1.43 1.66 1.36 95% CI Upper limit of 8.61 9.26 6.23 5.62 6.77 5.78 6.98 8.42 6.38 95% CI

TABLE 21.5 Comparison of marker levels and the area under the ROC curve (AUC) in EDTA samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Non-persistent Non-persistent Cohort Persistent Cohort Cohort Persistent Cohort Cohort Persistent Cohort sCr or UO Median 37100 58200 41000 59900 42700 60600 Average 42700 61000 45100 63100 45600 63700 Stdev 21300 26400 21200 27200 20800 27700 p (t-test) 5.2E−5 2.1E−5 1.5E−5 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 47 103 65 85 70 80 sCr only Median 37300 57200 41800 59100 43200 59100 Average 43700 61100 45900 62700 46600 63300 Stdev 21700 26500 21700 27300 21200 28000 p (t-test) 9.7E−5 7.1E−5 6.6E−5 Min 18800 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 50 100 66 84 72 78 UO only Median 47200 63600 47000 64000 47400 64500 Average 48300 66200 48400 67000 48700 67800 Stdev 20600 30600 20800 30700 20600 31500 p (t-test) 3.7E−5 1.9E−5 1.8E−5 Min 17100 15600 17100 15600 17100 15600 Max 123000 141000 123000 141000 123000 141000 n (Patient) 92 57 95 54 99 50 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.71 0.68 0.71 0.69 0.68 0.70 0.68 0.68 SE 0.042 0.042 0.046 0.042 0.043 0.047 0.042 0.043 0.048 p Value 8.5E−8 8.2E−7 1.3E−4 7.3E−7 7.7E−6 7.8E−5 1.3E−6 2.5E−5 1.5E−4 nCohort 47 50 92 65 66 95 70 72 99 Non-persistent nCohort Persistent 103 100 57 85 84 54 80 78 50 Cutoff Quartile 2 34800 34800 34700 34800 34800 34700 34800 34800 34700 Sensitivity 82% 82% 81% 82% 82% 81% 82% 82% 80% Specificity 40% 40% 28% 35% 35% 28% 34% 33% 27% Cutoff Quartile 3 52000 52000 51800 52000 52000 51800 52000 52000 51800 Sensitivity 60% 60% 65% 64% 63% 67% 64% 63% 66% Specificity 72% 70% 59% 68% 67% 59% 66% 64% 58% Cutoff Quartile 4 70700 70700 70500 70700 70700 70500 70700 70700 70500 Sensitivity 32% 32% 40% 35% 35% 41% 38% 37% 42% Specificity 89% 88% 84% 88% 86% 83% 89% 88% 83% OR Quartile 2 3.00 3.04 1.65 2.56 2.46 1.75 2.46 2.29 1.50 p Value 0.0050 0.0043 0.22 0.015 0.019 0.18 0.020 0.033 0.33 Lower limit of 95% CI 1.39 1.42 0.741 1.20 1.16 0.770 1.15 1.07 0.659 Upper limit of 95% CI 6.46 6.51 3.66 5.44 5.23 3.96 5.25 4.88 3.41 OR Quartile 3 3.95 3.50 2.63 3.65 3.42 2.87 3.37 2.99 2.63 p Value 3.3E−4 7.1E−4 0.0056 2.0E−4 3.7E−4 0.0031 3.9E−4 0.0012 0.0073 Lower limit of 95% CI 1.87 1.69 1.33 1.85 1.74 1.43 1.72 1.54 1.30 Upper limit of 95% CI 8.38 7.23 5.21 7.22 6.73 5.77 6.60 5.81 5.35 OR Quartile 4 3.96 3.45 3.47 3.89 3.34 3.39 4.65 4.14 3.49 p Value 0.0079 0.011 0.0014 0.0021 0.0046 0.0017 4.9E−4 8.6E−4 0.0014 Lower limit of 95% CI 1.43 1.33 1.62 1.64 1.45 1.58 1.96 1.80 1.62 Upper limit of 95% CI 10.9 8.93 7.47 9.22 7.69 7.29 11.0 9.56 7.52

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims. 

1-111. (canceled)
 112. A method of treating a subject, the method comprising: (a) performing an assay method to detect a level of Follistatin-related protein 3 in a body fluid sample obtained from a subject, wherein the Follistatin-related protein 3 comprises amino acid residues 27-263 of SEQ ID NO: 1, and wherein the sample has an elevated level of the Follistatin-related protein 3 as compared to a predetermined threshold value; (b) determining that the subject is at an increased risk of a persistent RIFLE I (Injury) acute kidney injury or a persistent RIFLE F (Failure) acute kidney injury based on the level of the Follistatin-related protein 3 in the sample; and (c) treating the subject determined to be at an increased risk as in step (b) by delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, monitoring and optimizing hemodynamics and fluid administration, and adjusting dosing of renally excreted substances.
 113. The method of claim 112, wherein the Follistatin-related protein 3 consists of amino acid residues 27-263 of SEQ ID NO:
 1. 114. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistent RIFLE I acute kidney injury or the persistent RIFLE F acute kidney injury that persists for a period of 24 hours or more after the time the sample is obtained from the subject.
 115. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistent RIFLE I acute kidney injury or the persistent RIFLE F acute kidney injury that persists for a period of 48 hours or more after the time the sample is obtained from the subject.
 116. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistent RIFLE I acute kidney injury or the persistent RIFLE F acute kidney injury that persists for a period of 72 hours or more after the time the sample is obtained from the subject.
 117. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of a future persistence of a RIFLE I acute kidney injury or a future persistence of a RIFLE F acute kidney injury that starts after the time the sample is obtained.
 118. The method of claim 117, wherein the future persistence comprises a persistence period that starts within 24 hours after the sample is obtained.
 119. The method of claim 117, wherein the future persistence comprises a persistence period that starts within 48 hours after the sample is obtained.
 120. The method of claim 117, wherein the future persistence comprises a persistence period that starts within 72 hours after the sample is obtained.
 121. The method of claim 112, wherein the persistent RIFLE I acute kidney injury or the persistent RIFLE F acute kidney injury comprises a persistence period that starts at the time the sample is obtained.
 122. The method of claim 121, wherein the determining comprises determining that the subject has an increased likelihood of the persistent RIFLE I acute kidney injury or the persistent RIFLE F acute kidney injury that persists for a period of 24, 48, or 72 hours after the time the sample is obtained from the subject.
 123. The method of claim 112, wherein the determining comprises determining that the subject is at an increased risk of a persistent RIFLE F acute kidney injury within 48 hours after the sample is obtained, wherein the duration of the persistent RIFLE F acute kidney injury comprises a 72-hour period with a minimum RIFLE stage of RIFLE F.
 124. The method of claim 123, wherein the duration of the persistent RIFLE F acute kidney injury comprises a 7-day period with a minimum RIFLE stage of RIFLE F.
 125. The method of claim 112, wherein the method further comprises contacting the sample with a binding reagent which binds to the Follistatin-related protein 3, and generating an assay result indicative of binding of the Follistatin-related protein 3 to the binding reagent.
 126. The method of claim 125, wherein the binding reagent is an antibody.
 127. The method of claim 112, further comprising performing an assay method to detect a level of one or more of Basigin, Cathepsin B, and Tenascin in the sample, wherein the Basigin comprises amino acid residues 22-385 of SEQ ID NO: 4, amino acid residues 22-23 and 140-385 of SEQ ID NO: 4, amino acid residues 210-385 of SEQ ID NO: 4, and/or amino acid residues 192-385 of SEQ ID NO: 4, the Cathepsin B comprises amino acid residues 18-79 of SEQ ID NO: 2, amino acid residues 80-333 of SEQ ID NO: 2, amino acid residues 80-126 of SEQ ID NO: 2, amino acid residues 129-133 of SEQ ID NO: 2, and/or amino acid residues 334-339 of SEQ ID NO: 2, the Tenascin comprises amino acid residues 23-2201 of SEQ ID NO: 3, amino acid residues 23-1071, 1436-1526, and 1618-2201 of SEQ ID NO: 3, amino acid residues 23-1071 and 1436-2201 of SEQ ID NO: 3, amino acid residues 23-1526 and 1618-2201 of SEQ ID NO: 3, amino acid residues 23-1071 and 1618-2201 of SEQ ID NO: 3, and/or amino acid residues 23-1071 and 1709-2201 of SEQ ID NO: 3, and wherein the subject has an elevated level of the one or more of Basigin, Cathepsin B, and Tenascin as compared to a predetermined threshold value.
 128. The method of claim 127, wherein the method further comprises contacting the sample with a binding reagent which binds to the Follistatin-related protein 3 and generating an assay result indicative of binding of the Follistatin-related protein 3 to the binding reagent, and contacting the sample with a binding reagent which binds to the one or more of Basigin, Cathepsin B, and Tenascin, and generating an assay result indicative of binding of the one or more of Basigin, the Cathepsin B, and the Tenascin to the binding reagent.
 129. The method of claim 128, wherein the binding agent is an antibody.
 130. The method of claim 112, wherein the body fluid sample is a urine sample, a blood sample, a plasma sample, or a serum sample.
 131. A method of treating a subject, the method comprising: (a) performing an assay method to detect a level of Follistatin-related protein 3 in a body fluid sample obtained from a subject, wherein the Follistatin-related protein 3 comprises amino acid residues 27-263 of SEQ ID NO: 1, and wherein the sample has an elevated level of the Follistatin-related protein 3 as compared to a predetermined threshold value; (b) determining that the subject is at an increased risk of a persistent RIFLE F (Failure) acute kidney injury within 48 hours of the time the sample is obtained, based on the level of the Follistatin-related protein 3 in the sample, wherein the duration of the persistent RIFLE F acute kidney injury comprises a 72-hour period with a minimum RIFLE stage of RIFLE F; and (c) treating the subject determined to be at an increased risk as in step (b) by delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, monitoring and optimizing hemodynamics and fluid administration, and adjusting dosing of renally excreted substances. 